Peptidyl prodrugs and linkers and stabilizers useful therefor

ABSTRACT

The present invention provides analogues of duocarmycins that are potent cytotoxins. Also provided are peptidyl and disulfide linkers that are cleaved in vivo. The linkers are of use in forming prodrugs and conjugates of the cytotoxins of the invention as well as other diagnostic and therapeutic moieties. The invention provides prodrugs and conjugates of the duocarmycin analogues with the linker arms of the invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/224,580, filed Sep. 12, 2005, which is a continuation ofU.S. patent application Ser. No. 10/161,234, filed May 31, 2002, issuedas U.S. Pat. No. 7,087,600, on Aug. 8, 2006, which claims priority ofU.S. Provisional Patent Application Nos. 60/295,196, filed May 31, 2001,60/295,259, filed May 31, 2001, 60/295,342, filed May 31, 2001, and60/304,908, filed Jul. 11, 2001. The disclosure of each of theprovisional applications is incorporated herein by reference in itsentirety for all purposes.

BACKGROUND OF THE INVENTION

Many therapeutic agents, particularly those that are especiallyeffective in cancer chemotherapy, often exhibit acute toxicity in vivo,especially bone marrow and mucosal toxicity, as well as chronic cardiacand neurological toxicity. Such high toxicity can limit theirapplications. Development of more and safer specific therapeutic agents,particularly antitumor agents, is desirable for greater effectivenessagainst tumor cells and a decrease in the number and severity of theside effects of these products (toxicity, destruction of non-tumorcells, etc.). Another difficulty with some existing therapeutic agentsis their less than optimal stability in plasma. Addition of functionalgroups to stabilize these compounds resulted in a significant loweringof the activity. Accordingly, it is desirable to identify ways tostabilize compounds while maintaining acceptable therapeutic activitylevels.

The search for more selective cytotoxic agents has been extremely activefor many decades, the dose limiting toxicity (i.e., the undesirableactivity of the cytotoxins on normal tissues) being one of the majorcauses of failures in cancer therapy. For example, CC-1065 and theduocarmycins are known to be extremely potent cytotoxins.

CC-1065 was first isolated from Streptomyces zelensis in 1981 by theUpjohn Company (Hanka et al., J. Antibiot. 31: 1211 (1978); Martin etal., J. Antibiot. 33: 902 (1980); Martin et al., J. Antibiot. 34: 1119(1981)) and was found to have potent antitumor and antimicrobialactivity both in vitro and in experimental animals (Li et al., CancerRes. 42: 999 (1982)). CC-1065 binds to double-stranded B-DNA within theminor groove (Swenson et al., Cancer Res. 42: 2821 (1982)) with thesequence preference of 5′-d(A/GNTTA)-3′ and 5′-d(AAAAA)-3′ and alkylatesthe N3 position of the 3′-adenine by its CPI left-hand unit present inthe molecule (Hurley et al., Science 226: 843 (1984)). Despite itspotent and broad antitumor activity, CC-1065 cannot be used in humansbecause it causes delayed death in experimental animals.

Many analogues and derivatives of CC-1065 and the duocarmycins are knownin the art. The research into the structure, synthesis and properties ofmany of the compounds has been reviewed. See, for example, Boger et al.,Angew. Chem. Int. Ed. Engl. 35: 1438 (1996); and Boger et al., Chem.Rev. 97: 787 (1997).

A group at Kyowa Hakko Kogya Co., Ltd. has prepared a number of CC-1065derivatives. See, for example, U.S. Pat. Nos. 5,101,038; 5,641,780;5,187,186; 5,070,092; 5,703,080; 5,070,092; 5,641,780; 5,101,038; and5,084,468; and published PCT application, WO 96/10405 and publishedEuropean application 0 537 575 A1. None of the patents or applicationsdisclose the strategy of enhancing the stability of the cytotoxins byforming cleaveable prodrugs.

The Upjohn Company (Pharmacia Upjohn) has also been active in preparingderivatives of CC-1065. See, for example, U.S. Pat. Nos. 5,739,350;4,978,757, 5,332,837 and 4,912,227. The issued U.S. patents do notdisclose or suggest that a prodrug strategy would be useful to improvethe in vivo stability or reduce the toxicity of the compounds disclosedin the patents.

Research has also focused on the development of new therapeutic agentswhich are in the form of prodrugs, compounds that are capable of beingconverted to drugs (active therapeutic compounds) in vivo by certainchemical or enzymatic modifications of their structure. For purposes ofreducing toxicity, this conversion is preferably confined to the site ofaction or target tissue rather than the circulatory system or non-targettissue. However, even prodrugs are problematic as many are characterizedby a low stability in blood and serum, due to the presence of enzymesthat degrade or activate the prodrugs before the prodrugs reach thedesired sites within the patient's body.

Therefore, in spite of the advances in the art, there continues to be aneed for the development of improved therapeutic agents for thetreatment of mammals and humans in particular, more specificallycytotoxins that exhibit high specificity of action, reduced toxicity,and improved stability in blood relative to known compounds of similarstructure. The instant invention addresses those needs.

SUMMARY OF THE INVENTION

The present invention relates to cytotoxins that are analogs of CC-1065and the duocarmycins. The present invention also provides linker armsthat are cleaved, for example, enzymatically or reductively in vivo,releasing an active drug moiety from the prodrug derivative thatincludes the linker arm. Furthermore, the invention includes conjugatesbetween the linker arms and the cytotoxins of the invention, andconjugates between the linker arms, the cytotoxin and a targeting agent,such as an antibody or a peptide.

The invention also relates to groups useful for stabilizing therapeuticagents and markers. The stabilizing groups are selected to limitclearance and metabolism of the therapeutic agent or marker by enzymesthat may be present in blood or non-target tissue and are furtherselected to limit transport of the agent or marker into the cells. Thestabilizing groups serve to block degradation of the agent or marker andmay also act in providing other physical characteristics of the agent ormarker. The stabilizing group may also improve the agent or marker'sstability during storage in either a formulated or non-formulated form.

In a first aspect, the invention provides a cytotoxic compound having astructure according to Formula I:

in which ring system A is a member selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl groups. The symbols E andG represent H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, a heteroatom, or a single bond. E and G areoptionally joined to form a ring system selected from substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl.

In an exemplary embodiment, ring system A is a substituted orunsubstituted phenyl ring. Ring system A is preferably substituted withone or more aryl group substituents as set forth in the definitionssection herein. In one preferred embodiment, the phenyl ring issubstituted with a CN moiety.

The curved line within the six-membered ring to which R³ is attachedindicates that the ring system may have one or more than one degree ofunsaturation at any position within the ring, and it may indicatearomaticity.

The symbol X represents a member that is selected from O, S and NR²³.R²³ is a member selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, and acyl.

The symbol R³ represents a member selected from (═O), SR¹¹, NHR¹¹ andOR¹¹, in which R¹¹ is H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, acyl, C(O)R¹², C(O)OR¹², C(O)NR¹²R¹³,C(O)OR¹², P(O)(OR¹²)₂, C(O)CHR¹²R¹³, C(O)OR¹², SR¹² or SiR¹²R¹³R¹⁴. Thesymbols R¹², R¹³, and R¹⁴ independently represent H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl, wherein R¹² and R¹³ together with thenitrogen atom to which they are attached are optionally joined to form asubstituted or unsubstituted heterocycloalkyl ring system having from 4to 6 members, optionally containing two or more heteroatoms.

R⁴ and R⁵ are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted arylalkyl, halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵,OC(O)NR¹⁵R¹⁶, OC(O)OR¹⁵, C(O)R¹⁵, OP(O)OR¹⁵OR¹⁶ and OR¹⁵. R¹⁵ and R¹⁶independently represent H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl, arylalkyl and substituted orunsubstituted peptidyl, wherein R¹⁵ and R¹⁶ together with the nitrogenatom to which they are attached are optionally joined to form asubstituted or unsubstituted heterocycloalkyl ring system having from 4to 6 members, optionally containing two or more heteroatoms.

R⁴, R⁵, R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ optionally contain one or morecleaveable groups within their structure. Exemplary cleaveable groupsinclude, but are not limited to peptides, amino acids and disulfides.

R⁶ is a single bond which is either present or absent. When R⁶ ispresent, R⁶ and R⁷ are joined to form a cyclopropyl ring. R⁷ is CH₂—X¹or —CH₂—. When R⁷ is —CH₂— it is a component of the cyclopropane ring.The symbol X¹ represents a leaving group. Those of skill will interpretcombinations of R⁶ and R⁷ in a manner that does not violate theprinciples of chemical valence.

In another aspect, the invention provides cleaveable linker arms thatinclude a group that is cleaved by an enzyme. The cleaveable linkergenerally imparts in vivo cleavability to the construct. Thus, thelinker may include one or more groups that will cleave in vivo, e.g., inthe blood stream at a rate which is enhanced relative to that ofconstructs which lack such groups. Also provided are conjugates of thelinker arms with therapeutic and diagnostic agents. The linkers areuseful to form prodrug analogs of therapeutic agents and to reversiblylink a therapeutic or diagnostic agent to a targeting agent, adetectable label, or a solid support. The linkers may be incorporatedinto complexes that include the cytotoxins of the invention. The linkershave the general formula set forth in Formula II:

In the formula above, the symbol E represents an enzymaticallycleaveable moiety (e.g., peptide, ester, etc.). The symbols R, R^(I),R^(II) and R^(III) represent members that include, for example, H,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, poly(ethylene glycol), acyl, a targeting agent, adetectable label. In a presently preferred embodiment, the oxygen of thecarboxyl moiety is tethered to a moiety that is a detectable label, atherapeutic moiety or a solid support.

In yet a further aspect, the invention provides a cleaveable linker armthat is based upon a disulfide moiety. Thus, there is provided acompound having a structure according to Formula III:

The identities of the radicals represented by the symbols R, R^(I),R^(II), R^(III), R^(IIII), R^(v) and R^(vI) are as described for R,R^(I), R^(II) and R^(III) above.

Other aspects, advantages and objects of the invention will be apparentfrom review of the detailed description below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth exemplary cleaveable urethane linkers of the inventionconjugated to a cytotoxin.

FIG. 2 sets forth exemplary cytotoxins of the invention.

FIG. 3 sets forth exemplary cleaveable disulfide linkers of theinvention conjugated to a cytotoxin.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTSAbbreviations

As used herein, “Ala,” refers to alanine.

“Boc,” refers to t-butyloxycarbonyl.

“DDQ,” refers to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

As used herein, the symbol “E,” represents an enzymatically cleaveablegroup.

“EDCI” is 1-(3-dimethylaminopropyl-3-ethylcarbodiimide.

As used herein, “FMOC,” refers to 9-fluorenylmethyloxycarbonyl.

“Leu” is leucine.

The symbol “PMB,” refers to para-methoxybenzyl.

“TBAF,” refers to tetrabutylammonium fluoride.

The abbreviation “TBSO,” refers to t-butyldimethylsilyl ether.

“TFA,” refers to trifluoroacetic acid.

The symbol “Q,” refers to a therapeutic agent, diagnostic agent ordetectable label.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. Generally, enzymatic reactions andpurification steps are performed according to the manufacturer'sspecifications. The techniques and procedures are generally performedaccording to conventional methods in the art and various generalreferences (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses. The term “therapeuticagent” is intended to mean a compound that, when present in atherapeutically effective amount, produces a desired therapeutic effecton a mammal. For treating carcinomas, it is desirable that thetherapeutic agent also be capable of entering the target cell.

The term “cytotoxin” is intended to mean a therapeutic agent having thedesired effect of being cytotoxic to cancer cells. Exemplary cytotoxinsinclude, by way of example and not limitation, combretastatins,duocarmycins, the CC-1065 anti-tumor antibiotics, anthracyclines, andrelated compounds. Other cytotoxins include mycotoxins, ricin and itsanalgoues, calicheamycins, doxirubicin and maytansinoids.

The term “marker” is intended to mean a compound useful in thecharacterization of tumors or other medical condition, for example,diagnosis, progression of a tumor, and assay of the factors secreted bytumor cells. Markers are considered a subset of “diagnostic agents.”

The term “targeting group” is intended to mean a moiety that is (1) ableto direct the entity to which it is attached (e.g., therapeutic agent ormarker) to a target cell, for example to a specific type of tumor cellor (2) is preferentially activated at a target tissue, for example atumor. The targeting group can be a small molecule, which is intended toinclude both non-peptides and peptides. The targeting group can also bea macromolecule, which includes saccharides, lectins, receptors, ligandfor receptors, proteins such as BSA, antibodies, and so forth.

The term “cleaveable group” is intended to mean a moiety that isunstable in vivo. Preferably the “cleaveable group” allows foractivation of the marker or therapeutic agent by cleaving the marker oragent from the rest of the conjugate. Operatively defined, the linker ispreferably cleaved in vivo by the biological environment. The cleavagemay come from any process without limitation, e.g., enzymatic,reductive, pH, etc. Preferably, the cleaveable group is selected so thatactivation occurs at the desired site of action, which can be a site inor near the target cells (e.g., carcinoma cells) or tissues such as atthe site of therapeutic action or marker activity. Such cleavage isenzymatic and exemplary enzymatically cleaveable groups include naturalamino acids or peptide sequences that end with a natural amino acid, andare attached at their carboxyl terminus to the linker. While the degreeof cleavage rate enhancement is not critical to the invention, preferredexamples of cleaveable linkers are those in which at least about 10% ofthe cleaveable groups are cleaved in the blood stream within 24 hours ofadministration, most preferably at least about 35%. Preferred cleaveablegroups are peptide bonds, ester linkages, and disulfide linkages.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer. These termsalso encompass the term “antibody.”

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but functions in a mannersimilar to a naturally occurring amino acid. The term “unnatural aminoacid” is intended to represent the “D” stereochemical form of the twentynaturally occurring amino acids described above. It is furtherunderstood that the term unnatural amino acid includes homologues of thenatural amino acids, and synthetically modified forms of the naturalamino acids. The synthetically modified forms include, but are notlimited to, amino acids having alkylene chains shortened or lengthenedby up to two carbon atoms, amino acids comprising optionally substitutedaryl groups, and amino acids comprised halogenated groups, preferablyhalogenated alkyl and aryl groups. When attached to a linker orconjugate of the invention, the amino acid is in the form of an “aminoacid side chain”, where the carboxylic acid group of the amino acid hasbeen replaced with a keto (C(O)) group. Thus, for example, an alanineside chain is —C(O)—CH(NH₂)—CH₃, and so forth.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

The symbol

whether utilized as a bond or displayed perpendicular to a bondindicates the point at which the displayed moiety is attached to theremainder of the molecule, solid support, etc.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- and multivalentradicals, having the number of carbon atoms designated (i.e., C₁-C₁₀means one to ten carbons). Examples of saturated hydrocarbon radicalsinclude, but are not limited to, groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “alkyl,” unlessotherwise noted, is also meant to include those derivatives of alkyldefined in more detail below, such as “heteroalkyl.” Alkyl groups, whichare limited to hydrocarbon groups are termed “homoalkyl”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified, but notlimited, by —CH₂CH₂CH₂CH₂—, and further includes those groups describedbelow as “heteroalkylene.” Typically, an alkyl (or alkylene) group willhave from 1 to 24 carbon atoms, with those groups having 10 or fewercarbon atoms being preferred in the present invention. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si and S, and wherein the nitrogen, carbonand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S and Si maybe placed at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Examples include, but are not limited to, —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂,—S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,and —CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, suchas, for example, —CH₂—NH—OCH₃ and CH₂—O—Si(CH₃)₃. Similarly, the term“heteroalkylene” by itself or as part of another substituent means adivalent radical derived from heteroalkyl, as exemplified, but notlimited by, —CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. Forheteroalkylene groups, heteroatoms can also occupy either or both of thechain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino,alkylenediamino, and the like). The terms “heteroalkyl” and“heteroalkylene” encompass poly(ethylene glycol) and its derivatives(see, for example, Shearwater Polymers Catalog, 2001). Still further,for alkylene and heteroalkylene linking groups, no orientation of thelinking group is implied by the direction in which the formula of thelinking group is written. For example, the formula —C(O)₂R′-representsboth —C(O)₂R′— and —R′C(O)₂—.

The term “lower” in combination with the terms “alkyl” or “heteroalkyl”refers to a moiety having from 1 to 6 carbon atoms.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

In general, an “acyl substituent” is also selected from the group setforth above. As used herein, the term “acyl substituent” refers togroups attached to, and fulfilling the valence of a carbonyl carbon thatis either directly or indirectly attached to the polycyclic nucleus ofthe compounds of the present invention.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of substituted or unsubstituted “alkyl” and substituted orunsubstituted “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. The heteroatoms and carbonatoms of the cyclic structures are optionally oxidized.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” is mean to include, but not be limited to,trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, andthe like.

The term “aryl” means, unless otherwise stated, a substituted orunsubstituted polyunsaturated, aromatic, hydrocarbon substituent whichcan be a single ring or multiple rings (preferably from 1 to 3 rings)which are fused together or linked covalently. The term “heteroaryl”refers to aryl groups (or rings) that contain from one to fourheteroatoms selected from N, O, and S, wherein the nitrogen, carbon andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. A heteroaryl group can be attached to theremainder of the molecule through a heteroatom. Non-limiting examples ofaryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl,4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl,2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl,2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. “Aryl” and “heteroaryl” alsoencompass ring systems in which one or more non-aromatic ring systemsare fused, or otherwise bound, to an aryl or heteroaryl system.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl, and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generally referred to as “alkyl substituents”and “heteroalkyl substituents,” respectively, and they can be one ormore of a variety of groups selected from, but not limited to: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″,—NRSO₂R′, —CN and —NO₂ in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R′, R″, R′″ andR″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., arylsubstituted with 1-3 halogens, substituted or unsubstituted alkyl,alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present. When R′ and R″ areattached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 5-, 6-, or 7-membered ring. For example, —NR′R″is meant to include, but not be limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical, the arylsubstituents and heteroaryl substituents are generally referred to as“aryl substituents” and “heteroaryl substituents,” respectively and arevaried and selected from, for example: halogen, —OR′, ═O, ═NR′, ═N—OR′,—NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″,—OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, (C₁-C₈)alkyl and heteroalkyl,unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl,and (unsubstituted aryl)oxy-(C₁-C₄)alkyl. When a compound of theinvention includes more than one R group, for example, each of the Rgroups is independently selected as are each R′, R″, R′″ and R″″ groupswhen more than one of these groups is present.

Two of the aryl substituents on adjacent atoms of the aryl or heteroarylring may optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(q)—U—, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)—B—, wherein A and B are independently CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S) and silicon (Si).

The symbol “R” is a general abbreviation that represents a substituentgroup that is selected from substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted heterocyclyl groups.

The term “pharmaceutically acceptable salts” includes salts of theactive compounds which are prepared with relatively nontoxic acids orbases, depending on the particular substituents found on the compoundsdescribed herein. When compounds of the present invention containrelatively acidic functionalities, base addition salts can be obtainedby contacting the neutral form of such compounds with a sufficientamount of the desired base, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable base addition salts includesodium, potassium, calcium, ammonium, organic amino, or magnesium salt,or a similar salt. When compounds of the present invention containrelatively basic functionalities, acid addition salts can be obtained bycontacting the neutral form of such compounds with a sufficient amountof the desired acid, either neat or in a suitable inert solvent.Examples of pharmaceutically acceptable acid addition salts includethose derived from inorganic acids like hydrochloric, hydrobromic,nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Alsoincluded are salts of amino acids such as arginate and the like, andsalts of organic acids like glucuronic or galactunoric acids and thelike (see, for example, Berge et al., “Pharmaceutical Salts”, Journal ofPharmaceutical Science, 1977, 66, 1-19). Certain specific compounds ofthe present invention contain both basic and acidic functionalities thatallow the compounds to be converted into either base or acid additionsalts.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compounddiffers from the various salt forms in certain physical properties, suchas solubility in polar solvents, but otherwise the salts are equivalentto the parent form of the compound for the purposes of the presentinvention.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

The term “attaching moiety” or “moiety for attaching a targeting group”refers to a moiety which allows for attachment of a targeting group tothe linker. Typical attaching groups include, by way of illustration andnot limitation, alkyl, aminoalkyl, aminocarbonylalkyl, carboxyalkyl,hydroxyalkyl, alkyl-maleimide, alkyl-N-hydroxylsuccinimide,poly(ethylene glycol)-maleimide and poly(ethyleneglycol)-N-hydroxylsuccinimide, all of which may be further substituted.The linker can also have the attaching moiety be actually appended tothe targeting group.

As used herein, the term “leaving group” refers to a portion of asubstrate that is cleaved from the substrate in a reaction.

“Antibody” generally refers to a polypeptide comprising a frameworkregion from an immunoglobulin or fragments thereof that specificallybinds and recognizes an antigen. The recognized immunoglobulins includethe kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′2, a dimer of Fab whichitself is a light chain joined to VH-CH1 by a disulfide bond. TheF(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region, thereby converting the F(ab)′2 dimer intoan Fab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3^(rd) ed. 1993).While various antibody fragments are defined in terms of the digestionof an intact antibody, one of skill will appreciate that such fragmentsmay be synthesized de novo either chemically or by using recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv).

For preparation of monoclonal or polyclonal antibodies, any techniqueknown in the art can be used (see, e.g., Kohler & Milstein, Nature256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983); Coleet al., pp. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985)).

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to the beta subunits.When appropriately high titers of antibody to the immunogen areobtained, blood is collected from the animal and antisera are prepared.Further fractionation of the antisera to enrich for antibodies reactiveto the protein can be done if desired.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6: 511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art.

In a still further preferred embodiment, the antibody is a human orhumanized antibody. “Humanized” refers to a non-human polypeptidesequence that has been modified to minimize immunoreactivity in humans,typically by altering the amino acid sequence to mimic existing humansequences, without substantially altering the function of thepolypeptide sequence (see, e.g., Jones et al., Nature 321: 522-525(1986), and published UK patent application No. 8707252). A “human”antibody is composed entirely of polypeptide sequences from humanantibody genes and can be obtained, for example, by phage displaymethods or from mice genetically altered to contain human immunoglobingenes.

“Solid support,” as used herein refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. Solid supports useful in practicing thepresent invention can include groups that are activated or capable ofactivation to allow selected species to be bound to the solid support. Asolid support can also be a substrate, for example, a chip, wafer orwell, onto which an individual, or more than one compound, of theinvention is bound.

“Reactive functional group,” as used herein refers to groups including,but not limited to, olefins, acetylenes, alcohols, phenols, ethers,oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides,cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acidsisonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters,sulfites, enanines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds. Reactive functional groups alsoinclude those used to prepare bioconjugates, e.g., N-hydroxysuccinimideesters, maleimides and the like (see, for example, Hermanson,BIOCONJUGATE TECHNIQUES, Academic press, San Diego, 1996). Methods toprepare each of these functional groups are well known in the art andtheir application to or modification for a particular purpose is withinthe ability of one of skill in the art (see, for example, Sandler andKaro, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS, Academic Press, SanDiego, 1989).

The compounds of the invention are prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.) VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5^(TH)ED., Longman Scientific and Technical Ltd., Essex, 1991, pp. 809-816;and Heller, Acc. Chem. Res. 23: 128 (1990).

Cytotoxins

Many therapeutic agents, particularly those that are especiallyeffective in cancer chemotherapy, often exhibit acute toxicity in vivo,especially bone marrow and mucosal toxicity, as well as chronic cardiacand neurological toxicity. Such high toxicity can limit theirapplications. Development of more and safer specific therapeutic agents,particularly antitumor agents, is desirable for greater effectivenessagainst tumor cells and a decrease in the number and severity of theside effects of these products (toxicity, destruction of non-tumorcells, etc.).

The search for more selective cytotoxic agents has been extremely activefor many decades, the dose limiting toxicities (i.e., the undesirableactivity of the cytotoxins on normal tissues) being one of the majorcauses of failures in cancer therapy. For example, CC-1065 and theduocarmycins are known to be extremely potent cytotoxins. Numerousattempts have been made to evaluate analogs of these compounds; however,most have been shown to exhibit undesirable toxicity at therapeuticdoses. Accordingly, the goal has been to improve the specificity ofanti-tumor agents for increased effectiveness against tumor cells, whiledecreasing adverse side effects, such as toxicity and the destruction ofnon-tumor cells.

Research has focused on the development of new therapeutic agents whichare in the form of prodrugs, compounds that are capable of beingconverted to drugs (active therapeutic compounds) in vivo by certainchemical or enzymatic modifications of their structure. For purposes ofreducing toxicity, this conversion is preferably confined to the site ofaction or target tissue rather than the circulatory system or non-targettissue. However, even prodrugs are problematic as many are characterizedby a low stability in blood and serum, due to the presence of enzymesthat degrade or activate the prodrugs before the prodrugs reach thedesired sites within the patient's body.

Therefore, in spite of the advances in the art, there continues to be aneed for the development of improved therapeutic agents for thetreatment of mammals and humans in particular, more specificallycytotoxins and related prodrugs that exhibit high specificity of action,reduced toxicity, and improved stability in blood relative to knowncompounds of similar structure.

In a first aspect, the invention provides a cytotoxic compound having astructure according to Formula I:

in which ring system A is a member selected from substituted orunsubstituted aryl substituted or unsubstituted heteroaryl andsubstituted or unsubstituted heterocycloalkyl groups. Exemplary ringsystems include phenyl and pyrrole.

The symbols E and G represent H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, a heteroatom, or a singlebond. E and G are optionally joined to form a ring system selected fromsubstituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl and substituted or unsubstituted heterocycloalkyl.

The symbol X represents a member selected from O, S and NR²³. R²³ is amember selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, and acyl.

The symbol R³ represents a member selected from (═O), SR¹¹, NHR¹¹ andOR¹¹, in which R¹¹ is H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, acyl, C(O)R¹², C(O)OR¹², C(O)NR¹²R¹³,C(O)OR¹², P(O)(OR¹²)₂, C(O)CHR¹²R¹³, C(O)OR¹², SR¹² or SiR¹²R¹³R¹⁴. Thesymbols R¹², R¹³, and R¹⁴ independently represent H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl, wherein R¹² and R¹³ together with thenitrogen atom to which they are attached are optionally joined to form asubstituted or unsubstituted heterocycloalkyl ring system having from 4to 6 members, optionally containing two or more heteroatoms. One or moreof R¹², R¹³, or R¹⁴ can include a cleaveable group within its structure.

R⁴ and R⁵ are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl,halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵, OC(O)NR¹⁵R¹⁶, OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵and OR¹⁵. R¹⁵ and R¹⁶ independently represent H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl andsubstituted or unsubstituted peptidyl, wherein R¹⁵ and R¹⁶ together withthe nitrogen atom to which they are attached are optionally joined toform a substituted or unsubstituted heterocycloalkyl ring system havingfrom 4 to 6 members, optionally containing two or more heteroatoms.

R⁴, R⁵, R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ optionally contain one or morecleaveable groups within their structure. Exemplary cleaveable groupsinclude, but are not limited to peptides, amino acids and disulfides.

In another exemplary embodiment, the invention provides a compoundaccording to Formula I, wherein at least one of R⁴, R⁵, R¹¹, R¹², R¹³,R¹⁵ and R¹⁶ comprises:

wherein R³⁰ is a member selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl. The symbols R³¹ andR³² independently represent H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl and substituted or unsubstituted heteroaryl, or R³¹ and R³²together are:

R³³ and R³⁴ independently represent H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. Thesymbol R³⁵ represents substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl or NR³⁶. R³⁶ is a member selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. X⁵ is O or NR³⁷ wherein R³⁷ is a member selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. In yet a further embodiment, at least one of R³³ and R³⁴ isselected from L⁵X⁶, wherein the identity of “L” and “X” is generally asdescribed herein.

In an exemplary embodiment, at least one of R³¹, R³², R³³ and R³⁴ in thestructure above, is an aryl or heteroaryl moiety that is substitutedwith a moiety that includes a protected or unprotected reactivefunctional group, a targeting agent or a detectable label.

In a still further exemplary embodiment, at least one of R⁴, R⁵, R¹¹,R¹², R¹³, R¹⁵ and R¹⁶ bears a reactive group appropriate for conjugatingthe compound according to Formula I to another molecule. In a furtherexemplary embodiment, R⁴, R⁵, R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ areindependently selected from substituted alkyl and substitutedheteroalkyl and have a reactive functional group at the free terminus ofthe alkyl or heteroalkyl moiety. One or more of R⁴, R⁵, R¹¹, R¹², R¹³,R¹⁵ and R¹⁶ may be conjugated to another species, e.g, targeting agent,detectable label, solid support, etc.

As will be apparent from the discussion herein, when at least one of R¹⁵and R¹⁶ is a reactive functional group, that group can be a component ofa bond between the compound according to Formula I and another molecule.In an exemplary embodiment in which at least one of R¹⁵ and R¹⁶ is alinkage between the compound of Formula I and another species, at leastone of R¹⁵ and R¹⁶ is a moiety that is cleaved by an enzyme.

In a further exemplary embodiment, at least one of R⁴ and R⁵ is:

In the formula above, the symbols X² and Z¹ represent membersindependently selected from O, S and NR²³. The groups R¹⁷ and R¹⁸ areindependently selected from H, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocycloalkyl, halogen, NO₂, NR¹⁹R²⁰, NC(O)R¹⁹,OC(O)NR¹⁹, OC(O)OR¹⁹, C(O)R¹⁹, SR¹⁹ or OR¹⁹.

The symbols R¹⁹ and R²⁰ independently represent substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted peptidyl, wherein R¹⁹ and R²⁰ together with thenitrogen atom to which they are attached are optionally joined to form asubstituted or unsubstituted heterocycloalkyl ring system having from 4to 6 members, optionally containing two or more heteroatoms, with theproviso that when Z¹ is NH, both R¹⁷ and R¹⁸ are not H, and R¹⁷ is notNH₂. Throughout the present specification, the symbols R¹⁹ and R²⁰ alsoencompass the groups set forth for R⁴ and R⁵. Thus, for example, it iswithin the scope of the present invention to provide compounds havingtwo or more of the fused phenyl-heterocyclic ring systems set forthimmediately above linked in series, or a fused ring in combination witha linker. Moreover, in those embodiments in which a linker is present,the linker may be present as an R⁴ or R⁵ substituent or as an R¹⁷ or R¹⁸substituent.

R⁶ is a single bond which is either present or absent. When R⁶ ispresent, R⁶ and R⁷ are joined to form a cyclopropyl ring. R⁷ is CH₂—XIor —CH₂—. When R⁷ is —CH₂— it is a component of the cyclopropane ring.The symbol X¹ represents a leaving group. The combinations of R⁶ and R⁷are interpreted in a manner that does not violate the principles ofchemical valence.

The curved line within the six-membered ring indicates that the ring mayhave one or more degree of unsaturation, and it may be aromatic. Thus,ring structures such as those set forth below, and related structures,are within the scope of Formula I:

In an exemplary embodiment, ring system A is a substituted orunsubstituted phenyl ring. Ring system A is preferably substituted withone or more aryl group substituents as set forth in the definitionssection herein. In one preferred embodiment, the phenyl ring issubstituted with a CN moiety.

In another exemplary embodiment, the invention provides a compoundhaving a structure according to Formula IV:

In this embodiment, the identities of the radicals R³, R⁴, R⁵, R⁶, R⁷and X are substantially as described above. The symbol Z is a memberindependently selected from O, S and NR²³. The symbol R²³ represents amember selected from H, substituted or unsubstituted alkyl, substitutedor unsubstituted heteroalkyl, and acyl. When both X and Z are NR²³, eachR²³ is independently selected. The symbol R¹ represents H, substitutedor unsubstituted lower alkyl, or C(O)R⁸. R⁸ is a member selected fromNR⁹R¹⁰, NR⁹NHR¹⁰ and OR⁹. R⁹, and R¹⁰ are independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl. The radical R² is H, or substituted or unsubstituted loweralkyl. It is generally preferred that when R² is substituted alkyl, itis other than perfluoroalkyl, e.g., CF₃.

As discussed above, X¹ may be a leaving group. Useful leaving groupsinclude, but are not limited to, halides, azides, sulfonic esters (e.g.,alkylsulfonyl, arylsulfonyl), oxonium ions, alkyl perchlorates,ammonioalkanesulfonate esters, alkylfluorosulfonates and fluorinatedcompounds (e.g., triflates, nonaflates, tresylates) and the like. Thechoice of these and other leaving groups appropriate for a particularset of reaction conditions is within the abilities of those of skill inthe art (see, for example, March J, ADVANCED ORGANIC CHEMISTRY, 2ndEdition, John Wiley and Sons, 1992; Sandler S R, Karo W, ORGANICFUNCTIONAL GROUP PREPARATIONS, 2nd Edition, Academic Press, Inc., 1983;and Wade L G, COMPENDIUM OF ORGANIC SYNTHETIC METHODS, John Wiley andSons, 1980).

In an exemplary embodiment R¹ is an ester moiety, such as CO₂CH₃. In afurther exemplary embodiment, R² is a lower alkyl group, which may besubstituted or unsubstituted. A presently preferred lower alkyl group isCH₃. In a still further embodiment, R¹ is CO₂CH₃, and R² is CH₃.

In yet another exemplary embodiment, R⁴ and R⁵ are members independentlyselected from H, halogen, NH₂, O(CH₂)₂N(Me)₂ and NO₂. R⁴ and R⁵ arepreferably not H or OCH₃.

In yet another exemplary embodiment, the invention provides compoundshaving a structure according to Formulae V and VI:

In the Formulae above, X is preferably O; and Z is preferably O.

The compounds according to Formula I, may also include peptidyl linkersas a substituent. The linker may be located at any desired position onthe compound. In an exemplary embodiment, at least one of R⁴, R⁵, R¹¹,R¹², R¹³, R¹⁵ and R¹⁶ has a structure according to Formula VII:

In the discussion that follows, the linker according to Formula VII isexemplified as being R¹¹. The focus of the discussion is in the interestof clarity only, and it will be apparent to those of skill that thelinker could be at any position of the compounds of the invention.

In Formula VII, the symbol X³ represents a protected or unprotectedreactive functional group, a detectable label or a targeting agent. Thegroups L¹ and L² are linkers selected from substituted or unsubstitutedalkyl and substituted or unsubstituted heteroalkyl groups. Exemplarylinkers, L¹ and L², comprises a poly(ethylene glycol) moiety. Thelinkers are either present or absent, thus, q and v are integersindependently selected from 0 and 1. The symbols AA¹, AA^(b) andAA^(b+1) represent either natural and unnatural α-amino acids. Thedashed line between AA¹ and AA^(b) indicates that any number of aminoacids may be intermediate to the two recited species. In an exemplaryembodiment, the total number of amino acids within the parenthesis (“b”)is from about 0 to about 20. In a further exemplary embodiment, “b” isan integer from about 1 to about 5.

An exemplary linker according to Formula VII is set forth in FormulaVIII:

in which, the symbols R²¹ and R²² independently represent substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocycloalkyl, detectablelabels and targeting agents. The groups R¹², and R²⁵ are independentlyselected from H, substituted or unsubstituted lower alkyl, an amino acidside chain, detectable labels, and targeting agents.

The amino acid portion of the structure, represented by AA¹, AA^(b) andAA^(b+1) is substantially similar to that of Formula VII.

In another embodiment, the compounds according to Formula I include alinker having a structure according to Formula IX:

in which X⁴ is a member selected from protected reactive functionalgroups, unprotected reactive functional groups, detectable labels andtargeting agents. L³ is a linker selected from substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl groups,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl. L⁴ is a linkerselected from substituted or unsubstituted alkyl and substituted orunsubstituted heteroalkyl groups, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedcycloalkyl. The symbols p and t represent integers independentlyselected from 0 and 1.

The linker according to Formula IX may be substituted onto any site ofthe molecule according to Formula I. In an exemplary embodiment, thelinker according to Formula IX is a member selected from R⁴, R⁵, R¹¹,R¹², R¹³, R¹⁵ and R¹⁶. Those of skill will appreciate that the linkermay also be a component of one or more of R¹⁷ or R¹⁵, or similar sitesin higher homologues of the compounds according to Formula I.

An exemplary linker according to Formula IX, is set forth in Formula X:

In Formula X, R²⁷ and R²⁸ are members independently selected from H,substituted or unsubstituted lower alkyl, amino acid side chains,detectable labels and targeting agents. The symbol “s” represents aninteger that can be selected to provide a linker of any desired length.Presently preferred are linkers in which “s” is an integer from 0 to 6,more preferably between 1 and 5.

In yet another exemplary embodiment, the invention provides moleculesaccording to Formula I, that are substituted with one or more linkersthat include a cleaveable disulfide moiety within their structure suchas that set forth in Formula XI:

in which X⁴ is a member selected from protected reactive functionalgroups, unprotected reactive functional groups, detectable labels andtargeting agents. L³ is a linker selected from substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl groups.L is a linker selected from substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl groups. The symbols p and trepresent integers independently selected from 0 and 1.

In an exemplary embodiment according to Formula XI, the linker L⁴ is asubstituted or unsubstituted ethylene moiety.

The group, X⁴ is a member selected from R²⁹, COOR²⁹, C(O)NR²⁹, andC(O)NNR²⁹ wherein R²⁹ is a member selected from substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted heteroaryl.

In yet another exemplary embodiment, R²⁹ is a member selected from H;OH; NHNH₂;

wherein R³⁰ represents substituted or unsubstituted alkyl terminatedwith a reactive functional group, substituted or unsubstitutedheteroaryl terminated with a functional group and -(L³)_(p)X⁴, whereineach L³, X⁴ and p are independently selected.

Also within the scope of the present invention are compounds of theinvention that are poly- or multi-valent species, including, forexample, species such as dimers, trimers, tetramers and higher homologsof the compounds of the invention or reactive analogues thereof. Thepoly- and multi-valent species can be assembled from a single species ormore than one species of the invention. For example, a dimeric constructcan be “homo-dimeric” or “heterodimeric.” Moreover, poly- andmulti-valent constructs in which a compound of the invention or areactive analogue thereof, is attached to an oligomeric or polymericframework (e.g., polylysine, dextran, hydroxyethyl starch and the like)are within the scope of the present invention. The framework ispreferably polyfunctional (i.e., having an array of reactive sites forattaching compounds of the invention). Moreover, the framework can bederivatized with a single species of the invention or more than onespecies of the invention.

Moreover, the present invention includes compounds which arefunctionalized to afford compounds having water-solubility that isenhanced relative to analogous compounds that are not similarlyfunctionalized. Thus, any of the substituents set forth herein can bereplaced with analogous radicals that have enhanced water solubility.For example, it is within the scope of the invention to, for example,replace a hydroxyl group with a diol, or an amine with a quaternaryamine, hydroxy amine or similar more water-soluble moiety. In apreferred embodiment, additional water solubility is imparted bysubstitution at a site not essential for the activity towards the ionchannel of the compounds set forth herein with a moiety that enhancesthe water solubility of the parent compounds. Methods of enhancing thewater-solubility of organic compounds are known in the art. Such methodsinclude, but are not limited to, functionalizing an organic nucleus witha permanently charged moiety, e.g., quaternary ammonium, or a group thatis charged at a physiologically relevant pH, e.g., carboxylic acid,amine. Other methods include, appending to the organic nucleus hydroxyl-or amine-containing groups, e.g., alcohols, polyols, polyethers, and thelike. Representative examples include, but are not limited to,polylysine, polyethyleneimine, poly(ethyleneglycol) andpoly(propyleneglycol). Suitable functionalization chemistries andstrategies for these compounds are known in the art. See, for example,Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS, ACSSymposium Series Vol. 469, American Chemical Society, Washington, D.C.,1991.

Exemplary cytotoxins of the invention are set forth in FIG. 2.

Prodrugs and Cleaveable Linkers

In addition to the linkers explicitly exemplified in the section aboveas being attached to cytotoxins of the invention, the present inventionalso provides cleaveable linker arms that are appropriate for attachmentto essentially any molecular species. The linker arm aspect of theinvention is exemplified herein by reference to their attachment to atherapeutic moiety. It will, however, be readily apparent to those ofskill in the art that the linkers can be attached to diverse speciesincluding, but not limited to, diagnostic agents, analytical agents,biomolecules, targeting agents, detectable labels and the like.

In one aspect, the present invention relates to linkers that are usefulto attach targeting groups to therapeutic agents and markers. In anotheraspect, the invention provides linkers that impart stability tocompounds, reduce their in vivo toxicity, or otherwise favorably affecttheir pharmacokinetics, bioavailability and/or pharmacodynamics. It isgenerally preferred that in such embodiments, the linker is cleaved,releasing the active drug, once the drug is delivered to its site ofaction. Thus, in one embodiment of the invention, the linkers of theinvention are traceless, such that once removed from the therapeuticagent or marker (such as during activation), no trace of the linker'spresence remains.

In another embodiment of the invention, the linkers are characterized bytheir ability to be cleaved at a site in or near the target cell such asat the site of therapeutic action or marker activity. Such cleavage ispreferably enzymatic in nature. This feature aids in reducing systemicactivation of the therapeutic agent or marker, reducing toxicity andsystemic side effects.

The linkers also serve to stabilize the therapeutic agent or markeragainst degradation while in circulation. This feature provides asignificant benefit since such stabilization results in prolonging thecirculation half-life of the attached agent or marker. The linker alsoserves to attenuate the activity of the attached agent or marker so thatthe conjugate is relatively benign while in circulation and has thedesired effect, for example is toxic, after activation at the desiredsite of action. For therapeutic agent conjugates, this feature of thelinker serves to improve the therapeutic index of the agent.

The stabilizing groups are preferably selected to limit clearance andmetabolism of the therapeutic agent or marker by enzymes that may bepresent in blood or non-target tissue and are further selected to limittransport of the agent or marker into the cells. The stabilizing groupsserve to block degradation of the agent or marker and may also act inproviding other physical characteristics of the agent or marker. Thestabilizing group may also improve the agent or marker's stabilityduring storage in either a formulated or non-formulated form.

Ideally, the stabilizing group is useful to stabilize a therapeuticagent or marker if it serves to protect the agent or marker fromdegradation when tested by storage of the agent or marker in human bloodat 37° C. for 2 hours and results in less than 20%, preferably less than2%, cleavage of the agent or marker by the enzymes present in the humanblood under the given assay conditions.

The present invention also relates to conjugates containing theselinkers. More particularly, the invention relates to prodrugs that maybe used for the treatment of disease, especially for cancerchemotherapy. Specifically, use of the linkers described herein providefor prodrugs that display a high specificity of action, a reducedtoxicity, and an improved stability in blood relative to prodrugs ofsimilar structure.

Thus, there is provided a linker may contain any of a variety of groupsas part of its chain which will cleave in vivo, e.g., in the bloodstream at a rate which is enhanced relative to that of constructs whichlack such groups. Also provided are conjugates of the linker arms withtherapeutic and diagnostic agents. The linkers are useful to formprodrug analogs of therapeutic agents and to reversibly link atherapeutic or diagnostic agent to a targeting agent, a detectablelabel, or a solid support. The linkers may be incorporated intocomplexes that include the cytotoxins of the invention.

In one embodiment, the invention provides linkers that have the generalformula set forth in Formula II:

In the formula above, the symbol E represents an enzymaticallycleaveable moiety (e.g., peptide, disulfide, ester, etc.). The symbolsR, R^(I), R^(II) and R^(III) represent members that include, forexample, H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, poly(ethyleneglycol), acyl, a targetingagent, or a detectable label. In an exemplary embodiment, the carbonylmoiety is tethered to a moiety that is a detectable label, a therapeuticmoiety or a solid support. The carbonyl moiety may be further attachedto an oxygen, sulfur, nitrogen or carbon at the position where thefragment is truncated. In a further exemplary embodiment, the carbonylmoiety is a component of a urethane. The oxygen tethered to the carbonylmoiety is attached to a targeting agent, cytotoxin, solid support or thelike.

Peptide-Based Linkers

In an exemplary embodiment, the enzymatically cleaveable group is anamino acid or peptide sequence ending with an amino acid attached at itscarboxyl terminus to the remainder of the linker. Presently preferredamino acids or peptides are those that are tumor activated. Thetumor-activated peptides are enzymatically cleaveable groups that arespecifically cleaved at a tumor site. Specific peptides that areactivated by specific enzymes associated with a selected tumor can beutilized; numerous such peptides are known in the art. Amino acids usedin the linker can be either natural or unnatural amino acids. In apreferred embodiment, at least one amino acid in the sequence is anatural amino acid. An exemplary preparation of a linker thatincorporates an amino acid moiety is set forth in Scheme 1, detailingthe conjugation of Combrestatin to a linker of the invention.

In Scheme 1, the EDCI mediated dehydrative coupling of a primary aminewith t-Boc protected leucine provides the protected leucine-amineconjugate. The conjugate is coupled to Combrestatin through thep-nitrophenylcarbonate activated Combrestatin and the product isdeprotected by cleavage of the t-Boc group with trifluoroacetic acid.

In another exemplary embodiment, the linker is a cyclic amino carbamateas set forth in Formula XII.

The radicals in the formula above are substantially the same as thosedescribed in the context of the linear linker. Two of R, R′, R″ and R′″together with the atoms to which they are attached form a substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, or substituted or unsubstitutedheterocycloalkyl moiety.

An exemplary synthetic route to a cyclic amino carbamate linker of theinvention is set forth in Scheme 2.

In Scheme 2, the p-methoxybenzyl protected phenyl diamine is coupled atthe unprotected aniline nitrogen with t-Boc protected leucine usingEDCI. The p-methoxybenzyl group is removed via the action of DDQ and thelinker arm is coupled to Cobrestatin using p-nitrophenyl carbonateactivated Combrestatin. The t-Boc group is removed with TFA, providingthe Combrestatin-linker complex.

In yet another embodiment, there is provided an amino carbamate benzylalcohol linker as set forth in Formula XIII below.

The identity of the radicals in the structure above is substantiallysimilar to those set forth above. R″″ represents any of the substituentsfor an aryl moiety discussed supra. When there is more than one R″″group, each of the R″″ groups is independently selected, and z is aninteger from 0 to 5.

An exemplary synthesis of an amino carbamate benzyl alcohol linker ofthe invention is set forth below in Scheme 3.

In Scheme 3, a t-butyldimethylsilyl O-protected phenyl alcohol with anactivated benzylic position is coupled to a therapeutic or diagnosticagent. The conjugate is treated with tetrabutylammonium fluoride,removing the t-butyldimethylsilyl protecting group. The free OH group isconverted to the active carbonate with p-nitrophenylchloroformate. Theactivated carbonate intermediate is used to couple protectedBocLeuNH(CH₂)₂NHEt to the OH group, forming the linker-agent conjugate.

Peptide Linker-Duocarmycin Conjugates

CC-1065 and the duocarmycins are known to be extremely potent antitumorcytotoxins, which exhibit undesirable toxicity at therapeutic dosages.By attaching a tumor activated peptide to the cytotoxin, systemictoxicity is reduced and therapeutic index is increased. Thus, thepresent invention also provides prodrug conjugates of the duocarmycins,as well as conjugates between a duocarmycin and a targeting or otheragent according to Formula I.

An exemplary synthetic scheme to a conjugate of the invention is setforth in Scheme 4. Additional synthetic routes are provided in theexamples appended hereto.

In Scheme 4, the cytotoxin is converted to the active carbonate withp-nitrophenylchloroformate and the activated derivative is contactedwith the FMOC-protected tumor activated peptide, forming a conjugate.The conjugate is treated with piperidine, removing the FMOC group andproviding the desired compound.

Many peptide sequences that are cleaved by enzymes in the serum, liver,gut, etc. are known in the art. An exemplary peptide sequence of theinvention includes a peptide sequence that is cleaved by a protease. Thefocus of the discussion that follows on the use of a protease-sensitivesequence is for clarity of illustration and does not serve to limit thescope of the present invention.

When the enzyme that cleaves the peptide is a protease, the linkergenerally includes a peptide containing a cleavage recognition sequencefor the protease. A cleavage recognition sequence for a protease is aspecific amino acid sequence recognized by the protease duringproteolytic cleavage. Many protease cleavage sites are known in the art,and these and other cleavage sites can be included in the linker moiety.See, e.g., Matayoshi et al. Science 247: 954 (1990); Dunn et al. Meth.Enzymol. 241: 254 (1994); Seidah et al. Meth. Enzymol. 244: 175 (1994);Thomberry, Meth. Enzymol. 244: 615 (1994); Weber et al. Meth. Enzymol.244: 595 (1994); Smith et al. Meth. Enzymol. 244: 412 (1994); Bouvier etal. Meth. Enzymol. 248: 614 (1995), Hardy et al., in AMYLOID PROTEINRECURSOR IN DEVELOPMENT, AGING, AND ALZHEIMERS DISEASE, ed. Masters etal. pp. 190-198 (1994).

Proteases have been implicated in cancer metastasis. Increased synthesisof the protease urokinase was correlated with an increased ability tometastasize in many cancers. Urokinase activates plasmin fromplasminogen, which is ubiquitously located in the extracellular spaceand its activation can cause the degradation of the proteins in theextracellular matrix through which the metastasizing tumor cells invade.Plasmin can also activate the collagenases thus promoting thedegradation of the collagen in the basement membrane surrounding thecapillaries and lymph system thereby allowing tumor cells to invade intothe target tissues (Dano, et al. Adv. Cancer. Res., 44: 139 (1985)).Thus, it is within the scope of the present invention to utilize as alinker a peptide sequence that is cleaved by urokinase.

The invention also provides the use of peptide sequences that aresensitive to cleavage by tryptases. Human mast cells express at leastfour distinct tryptases, designated α βI, βII, and βIII. These enzymesare not controlled by blood plasma proteinase inhibitors and only cleavea few physiological substrates in vitro. The tryptase family of serineproteases has been implicated in a variety of allergic and inflammatorydiseases involving mast cells because of elevated tryptase levels foundin biological fluids from patients with these disorders. However, theexact role of tryptase in the pathophysiology of disease remains to bedelineated. The scope of biological functions and correspondingphysiological consequences of tryptase are substantially defined bytheir substrate specificity.

Tryptase is a potent activator of pro-urokinase plasminogen activator(uPA), the zymogen form of a protease associated with tumor metastasisand invasion. Activation of the plasminogen cascade, resulting in thedestruction of extracellular matrix for cellular extravasation andmigration, may be a function of tryptase activation of pro-urokinaseplasminogen activator at the P4-P1 sequence of Pro-Arg-Phe-Lys (SEQ IDNO. 2) (Stack, et al., Journal of Biological Chemistry 269(13):9416-9419 (1994)). Vasoactive intestinal peptide, a neuropeptide that isimplicated in the regulation of vascular permeability, is also cleavedby tryptase, primarily at the Thr-Arg-Leu-Arg sequence (SEQ ID NO. 3)(Tam, et al., Am. J. Respir. Cell Mol. Biol. 3: 27-32 (1990)). TheG-protein coupled receptor PAR-2 can be cleaved and activated bytryptase at the Ser-Lys-Gly-Arg sequence (SEQ ID NO. 4) to drivefibroblast proliferation, whereas the thrombin activated receptor PAR-1is inactivated by tryptase at the Pro-Asn-Asp-Lys sequence (SEQ ID NO.5) (Molino et al., Journal of Biological Chemistry 272(7): 4043-4049(1997)). Taken together, this evidence suggests a central role fortryptase in tissue remodeling as a consequence of disease. This isconsistent with the profound changes observed in several mastcell-mediated disorders. One hallmark of chronic asthma and otherlong-term respiratory diseases is fibrosis and thickening of theunderlying tissues that could be the result of tryptase activation ofits physiological targets. Similarly, a series of reports have shownangiogenesis to be associated with mast cell density, tryptase activityand poor prognosis in a variety of cancers (Coussens et al., Genes andDevelopment 13(11): 1382-97 (1999)); Takanami et al., Cancer 88(12):2686-92 (2000); Toth-Jakatics et al., Human Pathology 31(8): 955-960(2000); Ribatti et al., International Journal of Cancer 85(2): 171-5(2000).

Methods are known in the art for evaluating whether a particularprotease cleaves a selected peptide sequence. For example, the use of7-amino-4-methyl coumarin (AMC) fluorogenic peptide substrates is awell-established method for the determination of protease specificity(Zimmerman, M., et al., (1977) Analytical Biochemistry 78:47-51).Specific cleavage of the anilide bond liberates the fluorogenic AMCleaving group allowing for the simple determination of cleavage ratesfor individual substrates. More recently, arrays (Lee, D., et al.,(1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72) andpositional-scanning libraries (Rano, T. A., et al., (1997) Chemistry andBiology 4:149-55) of AMC peptide substrate libraries have been employedto rapidly profile the N-terminal specificity of proteases by sampling awide range of substrates in a single experiment. Thus, one of skill mayreadily evaluate an array of peptide sequences to determine theirutility in the present invention without resort to undueexperimentation.

Disulfide Linkers

In yet a further aspect, the invention provides a cleaveable linker armthat is based upon a disulfide moiety. Thus, there is provided acompound having a structure according to Formula III:

The identities of the radicals represented by the symbols R, R^(I),R^(II), R^(III), R^(IIII), R^(v) and R^(vI) are as described for R,R^(I), R^(II) and R^(III) above.

In another embodiment, the invention provides a disulfide carbamatelinker such as that set forth in Formula XIV:

The identities of the radicals represented by the symbols R, R^(I),R^(II), R^(III), R^(IIII), R^(v) and R^(vI) are as described above.

As discussed above, the linkers of the invention can be used to formconjugates comprising a cytotoxin such as a combretastatin or aduocarmycin as the therapeutic agent. The duocarmycins are unstable inplasma. The linkers of the invention find particular utility instabilizing the duocarmycins while in circulation, and liberating theagent (activation) once at the desired site of action. In addition, inorder for the combretastatin or duocarmycin to regain maximum activityafter activation, both the linker and the targeting group are preferablycompletely removed. Therefore, in one embodiment of the invention, thelinkers are traceless linkers. Conjugates comprising a cytotoxin such asa duocarmycin as the therapeutic agent are also of particular interest.The linkers of the invention serve to stabilize duocarmycins incirculation and release an optimally potent cytotoxin after activationin or near the target cells. Since the cytotoxin is cleaved in or nearthe target cells, systemic toxicity due to random activation isdecreased. Further, the increase in stability in circulation alsoprovides for an increase in the half-life and overall effectiveness ofthe cytotoxin.

An exemplary route for preparing a disulfide linker arm-cytotoxinconjugate of the invention is set forth in Scheme 5.

In Scheme 5, an amine-protected sulfhydryl a is reacted with2,2′-dipyridyl disulfide b to form an amine-protected, activateddisulfide c. The activated disulfide is contacted with a carboxylicester bearing a free sulfhydryl d, eliminating pyridyl thiol and formingan amine-protected carboxylic ester that includes a disulfide moiety e.The methyl ester is cleaved by the action of LiOH to form thecorresponding carboxylic acid f. The carboxylic acid is coupled to aheterobifunctional PEG molecule that includes a maleimide group and anamine by the action of EDCI to form compound g. The PEG derivative iscontacted with an active carbonate of Combrestatin h to form conjugatei. If desired conjugate i can be attached to a targeting agent,detectable label or the like through the maleimide moiety.

In another embodiment, the invention provides a disulfide carbamatelinker in which the non-carbonyl oxygen of the urethane linkage isderived from an aryl group. A representative linker of the invention isset forth in Formula XV:

The identity of the radicals is essentially as described above. R^(vII)is a substituent on an aryl group as described in the definitionssection. The symbol w represents an integer from 0 to 4. When more thanone R^(vII) is present, each of the groups is independently selected.

An exemplary route to compounds according to Formula XV is set forth inScheme 6.

In Scheme 6, the TBS-alcohol protected benzyl bromide derivative a isreacted with Q-OH under alkylating conditions to form b. Compound b isdeprotected by the action of tetrabutylammonium fluoride, forming c,which is acylated with d, forming carbonate e. Carbonate e is reactedwith the heterobifunctional PEG derivative i from Scheme 5, supra. Theresulting PEG adduct f can be conjugated to another molecule through themaleimide moiety.

Disulfide Linker-Duocarmycin Conjugates

As discussed above, the disulfide linkers of the invention are alsouseful components for stabilizing therapeutic or diagnostic moieties invivo, forming prodrugs, and conjugating agents to species such astargeting agents, and detectable labels. Thus, in yet another aspect,the invention provides conjugates between a duocarmycin and a disulfidelinker of the invention according to Formula I. Scheme 7 provides afacile route to a conjugate of the invention.

In Scheme 7, a duocarmycin cytotoxin of the invention a converted to theactivated carbonate b with p-nitrophenylchloroformate. Compound b iscoupled to the heterobifunctional PEG linker from Scheme 5, formingcompound d, which may be subsequently coupled to an antibody through amaleimide-sulfhydryl coupling reaction to form conjugate e.

As discussed above, the therapeutic efficacy of certain toxic agents isdramatically improved by strategies that deliver the agent selectivelyto a desired site and/or maintain the agent in an essentially inactiveform until it is delivered to the desired site of action. The presentinvention also provides linker arms that operate according to theprinciple of targeting an agent to a selected site and/or inactivating abioactive agent until it reaches the desired site.

Thus, in certain embodiments, the invention provides conjugates of thecytotoxins set forth above, and of other agents as well, with linkerarms having efficacious properties. In one embodiment, the linker armconjugates a therapeutic or diagnostic moiety to an agent thatselectively delivers the moiety to a desired site in the body. Thelinker between the moiety and the targeting agent can be stable in vivo,or it can be cleaved. If the agent is cleaved, it is preferablypredominantly cleaved after it reaches the desired site of action.

In another embodiment, the invention provides linkers that do not tethera diagnostic or therapeutic moiety to another agent, but essentiallyinactivate the moiety until it reaches the desired site of activity;active species at the desired site of activity cleave the linker,preferably restoring the active form of the moiety. The strategyprovides a means to mitigate the systemic toxicity of many toxic, buthighly useful agents.

The urethane and disulfide linkers of the invention are exemplified incontext with their conjugation with representative duocarmycin analogsof the invention. See, FIG. 1 and FIG. 3, respectively.

Targeting Agents

The linker arms and cytotoxins of the invention can be linked totargeting agents that selectively deliver a payload to a cell, organ orregion of the body. Exemplary targeting agents such as antibodies (e.g.,chimeric, humanized and human), ligands for receptors, lectins,saccharides, antibodies, and the like are recognized in the art and areuseful without limitation in practicing the present invention. Othertargeting agents include a class of compounds that do not includespecific molecular recognition motifs include macromolecules such aspoly(ethylene glycol), polysaccharide, polyamino acids and the like,which add molecular mass to the cytotoxin. The additional molecular massaffects the pharmacokinetics of the cytotoxin, e.g., serum half-life.

In an exemplary embodiment, the invention provides a cytotoxin, linkeror cytotoxin-linker conjugate with a targeting agent that is abiomolecule, e.g, an antibody, receptor, peptide, lectin, saccharide,nucleic acid or a combination thereof. Routes to exemplary conjugates ofthe invention are set forth in the Schemes above.

Biomolecules useful in practicing the present invention can be derivedfrom any source. The biomolecules can be isolated from natural sourcesor can be produced by synthetic methods. Proteins can be naturalproteins or mutated proteins. Mutations can be effected by chemicalmutagenesis, site-directed mutagenesis or other means of inducingmutations known to those of skill in the art. Proteins useful inpracticing the instant invention include, for example, enzymes,antigens, antibodies and receptors. Antibodies can be either polyclonalor monoclonal. Peptides and nucleic acids can be isolated from naturalsources or can be wholly or partially synthetic in origin.

In those embodiments wherein the recognition moiety is a protein orantibody, the protein can be tethered to a SAM component or a spacer armby any reactive peptide residue available on the surface of the protein.In preferred embodiments, the reactive groups are amines orcarboxylates. In particularly preferred embodiments, the reactive groupsare the ε-amine groups of lysine residues. Furthermore, these moleculescan be adsorbed onto the surface of the substrate or SAM by non-specificinteractions (e.g., chemisorption, physisorption).

Recognition moieties which are antibodies can be used to recognizeanalytes which are proteins, peptides, nucleic acids, saccharides orsmall molecules such as drugs, herbicides, pesticides, industrialchemicals and agents of war. Methods of raising antibodies for specificmolecules are well-known to those of skill in the art. See, U.S. Pat.No. 5,147,786, issued to Feng et al. on Sep. 15, 1992; U.S. Pat. No.5,334,528, issued to Stanker et al. on Aug. 2, 1994; U.S. Pat. No.5,686,237, issued to Al-Bayati, M. A. S. on Nov. 11, 1997; and U.S. Pat.No. 5,573,922, issued to Hoess et al. on Nov. 12, 1996. Methods forattaching antibodies to surfaces are also art-known. See, Delamarche etal. Langmuir 12:1944-1946 (1996).

Targeting agents can be attached to the linkers of the invention by anyavailable reactive group. For example, peptides can be attached throughan amine, carboxyl, sulfhydryl, or hydroxyl group. Such a group canreside at a peptide terminus or at a site internal to the peptide chain.Nucleic acids can be attached through a reactive group on a base (e.g.,exocyclic amine) or an available hydroxyl group on a sugar moiety (e.g.,3′- or 5′-hydroxyl). The peptide and nucleic acid chains can be furtherderivatized at one or more sites to allow for the attachment ofappropriate reactive groups onto the chain. See, Chrisey et al. NucleicAcids Res. 24:3031-3039 (1996).

When the peptide or nucleic acid is a fully or partially syntheticmolecule, a reactive group or masked reactive group can be incorporatedduring the process of the synthesis. Many derivatized monomersappropriate for reactive group incorporation in both peptides andnucleic acids are know to those of skill in the art. See, for example,THE PEPTIDES: ANALYSIS, SYNTHESIS, BIOLOGY, Vol. 2: “Special Methods inPeptide Synthesis,” Gross, E. and Melenhofer, J., Eds., Academic Press,New York (1980). Many useful monomers are commercially available(Bachem, Sigma, etc.). This masked group can then be unmasked followingthe synthesis, at which time it becomes available for reaction with acomponent of a compound of the invention.

In another exemplary embodiment, the targeting moiety is attached to acompound of the invention via an inclusion complex. For example, acompound or linker of the invention can include a moiety such as acyclodextrin or modified cyclodextrin. Cyclodextrins are a group ofcyclic oligosaccharides produced by numerous microorganisms.Cyclodextrins have a ring structure which has a basket-like shape. Thisshape allows cyclodextrins to include many kinds of molecules into theirinternal cavity. See, for example, Szejtli, J., CYCLODEXTRINS AND THEIRINCLUSION COMPLEXES; Akademiai Klado, Budapest, 1982; and Bender et al.,CYCLODEXTRIN CHEMISTRY, Springer-Verlag, Berlin, 1978.

Cyclodextrins are able to form inclusion complexes with an array oforganic molecules including, for example, drugs, pesticides, herbicidesand agents of war. See, Tenjarla et al., J. Pharm. Sci. 87:425-429(1998); Zughul et al., Pharm. Dev. Technol. 3:43-53 (1998); and Alberset al., Crit. Rev. Ther. Drug Carrier Syst. 12:311-337 (1995).Importantly, cyclodextrins are able to discriminate between enantiomersof compounds in their inclusion complexes. Thus, in one preferredembodiment, the invention provides for the detection of a particularenantiomer in a mixture of enantiomers. See, Koppenhoefer et al. J.Chromatogr. A 793:153-164 (1998). Numerous routes for attachingcyclodextrins to other molecules are known in the art. See, for example,Yamamoto et al., J. Phys. Chem. B 101:6855-6860 (1997); and Sreenivasan,K. J. Appl. Polym. Sci. 60:2245-2249 (1996).

The cytotoxin-targeting agent conjugates of the invention are furtherexemplified by reference to an antisense oligonucleotide-cytotoxinconjugate. The focus on cytotoxin-oligonucleotide conjugates is forclarity of illustration and is not limiting of the scope of targetingagents to which the cytotoxins of the invention can be conjugated.

Exemplary nucleic acid targeting agents include aptamers, antisensecompounds, and nucleic acids that form triple helices. Typically, ahydroxyl group of a sugar residue, an amino group from a base residue,or a phosphate oxygen of the nucleotide is utilized as the neededchemical functionality to couple the nucleotide-based targeting agent tothe cytotoxin. However, one of skill in the art will readily appreciatethat other “non-natural” reactive functionalities can be appended to anucleic acid by conventional techniques. For example, the hydroxyl groupof the sugar residue can be converted to a mercapto or amino group usingtechniques well known in the art.

Aptamers (or nucleic acid antibody) are single- or double-stranded DNAor single-stranded RNA molecules that bind specific molecular targets.Generally, aptamers function by inhibiting the actions of the moleculartarget, e.g., proteins, by binding to the pool of the target circulatingin the blood. Aptamers possess chemical functionality and thus, cancovalently bond to cytotoxins, as described herein.

Although a wide variety of molecular targets are capable of formingnon-covalent but specific associations with aptamers, including smallmolecules drugs, metabolites, cofactors, toxins, saccharide-based drugs,nucleotide-based drugs, glycoproteins, and the like, generally themolecular target will comprise a protein or peptide, including serumproteins, kinins, eicosanoids, cell surface molecules, and the like.Examples of aptamers include Gilead's antithrombin inhibitor GS 522 andits derivatives (Gilead Science, Foster City, Calif.). See also, Macayaet al. Proc. Natl. Acad. Sci. USA 90:3745-9 (1993); Bock et al. Nature(London) 355:564-566 (1992) and Wang et al. Biochem. 32:1899-904 (1993).

Aptamers specific for a given biomolecule can be identified usingtechniques known in the art. See, e.g., Toole et al. (1992) PCTPublication No. WO 92/14843; Tuerk and Gold (1991) PCT Publication No.WO 91/19813; Weintraub and Hutchinson (1992) PCT Publication No.92/05285; and Ellington and Szostak, Nature 346:818 (1990). Briefly,these techniques typically involve the complexation of the moleculartarget with a random mixture of oligonucleotides. The aptamer-moleculartarget complex is separated from the uncomplexed oligonucleotides. Theaptamer is recovered from the separated complex and amplified. Thiscycle is repeated to identify those aptamer sequences with the highestaffinity for the molecular target.

For diseases that result from the inappropriate expression of genes,specific prevention or reduction of the expression of such genesrepresents an ideal therapy. In principle, production of a particulargene product may be inhibited, reduced or shut off by hybridization of asingle-stranded deoxynucleotide or ribodeoxynucleotide complementary toan accessible sequence in the mRNA, or a sequence within the transcriptthat is essential for pre-mRNA processing, or to a sequence within thegene itself. This paradigm for genetic control is often referred to asantisense or antigene inhibition. Additional efficacy is imparted by theconjugation to the nucleic acid of an alkylating agent, such as those ofthe present invention.

Antisense compounds are nucleic acids designed to bind and disable orprevent the production of the mRNA responsible for generating aparticular protein. Antisense compounds include antisense RNA or DNA,single or double stranded, oligonucleotides, or their analogs, which canhybridize specifically to individual mRNA species and preventtranscription and/or RNA processing of the mRNA species and/ortranslation of the encoded polypeptide and thereby effect a reduction inthe amount of the respective encoded polypeptide. Ching et al. Proc.Natl. Acad. Sci. U.S.A. 86:10006-10010 (1989); Broder et al. Ann. Int.Med. 113:604-618 (1990); Loreau et al. FEBS Letters 274:53-56 (1990);Holcenberg et al. WO91/11535; WO91/09865; WO91/04753; WO90/13641; WO91/13080, WO 91/06629, and EP 386563). Due to their exquisite targetsensitivity and selectivity, antisense oligonucleotides are useful fordelivering therapeutic agents, such as the cytotoxins of the inventionto a desired molecular target.

Others have reported that nucleic acids can bind to duplex DNA viatriple helix formation and inhibit transcription and/or DNA synthesis.Triple helix compounds (also referred to as triple strand drugs) areoligonucleotides that bind to sequences of double-stranded DNA and areintended to inhibit selectively the transcription of disease-causinggenes, such as viral genes, e.g., HIV and herpes simplex virus, andoncogenes, i.e., they stop protein production at the cell nucleus. Thesedrugs bind directly to the double stranded DNA in the cell's genome toform a triple helix and prevent the cell from making a target protein.See, e.g., PCT publications Nos. WO 92/10590, WO 92/09705, WO91/06626,and U.S. Pat. No. 5,176,996. Thus, the cytotoxins of the presentinvention are also conjugated to nucleic acid sequences that form triplehelices.

The site specificity of nucleic acids (e.g., antisense compounds andtriple helix drugs) is not significantly affected by modification of thephosphodiester linkage or by chemical modification of theoligonucleotide terminus. Consequently, these nucleic acids can bechemically modified; enhancing the overall binding stability, increasingthe stability with respect to chemical degradation, increasing the rateat which the oligonucleotides are transported into cells, and conferringchemical reactivity to the molecules. The general approach toconstructing various nucleic acids useful in antisense therapy has beenreviewed by van der Krol et al., Biotechniques 6:958-976 (1988) andStein et al. Cancer Res. 48:2659-2668 (1988). Therefore, in an exemplaryembodiment, the cytotoxins of the invention are conjugated to a nucleicacid by modification of the phosphodiester linkage.

Moreover, aptamers, antisense compounds and triple helix drugs bearingcytotoxins of the invention can also can include nucleotidesubstitutions, additions, deletions, or transpositions, so long asspecific hybridization to or association with the relevant targetsequence is retained as a functional property of the oligonucleotide.For example, some embodiments will employ phosphorothioate analogs whichare more resistant to degradation by nucleases than their naturallyoccurring phosphate diester counterparts and are thus expected to have ahigher persistence in vivo and greater potency (see, e.g., Campbell etal., J. Biochem. Biophys. Methods 20:259-267 (1990)). Phosphoramidatederivatives of oligonucleotides also are known to bind to complementarypolynucleotides and have the additional capability of accommodatingcovalently attached ligand species and will be amenable to the methodsof the present invention. See, for example, Froehler et al., NucleicAcids Res. 16(11):4831 (1988).

In some embodiments the aptamers, antisense compounds and triple helixdrugs will comprise O-methylribonucleotides (EP Publication No. 360609).Chimeric oligonucleotides may also be used (Dagle et al., Nucleic AcidsRes. 18: 4751 (1990)). For some applications, antisense oligonucleotidesand triple helix may comprise polyamide nucleic acids (Nielsen et al.,Science 254: 1497 (1991) and PCT publication No. WO 90/15065) or othercationic derivatives (Letsinger et al., J. Am. Chem. Soc. 110: 4470-4471(1988)). Other applications may utilize oligonucleotides wherein one ormore of the phosphodiester linkages has been substituted with anisosteric group, such as a 2-4 atom long internucleoside linkage asdescribed in PCT publication Nos. WO 92/05186 and 91/06556, or aformacetal group (Matteucci et al., J. Am. Chem. Soc. 113: 7767-7768(1991)) or an amide group (Nielsen et al., Science 254: 1497-1500(1991)).

In addition, nucleotide analogs, for example wherein the sugar or baseis chemically modified, can be employed in the present invention.“Analogous” forms of purines and pyrimidines are those generally knownin the art, many of which are used as chemotherapeutic agents. Anexemplary but not exhaustive list includes 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N⁶-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, .beta.-D-mannosylqueosine,5′methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N.sup.6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid (v), pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine. In addition, the conventional bases by halogenatedbases. Furthermore, the 2′-furanose position on the base can have anon-charged bulky group substitution. Examples of non-charged bulkygroups include branched alkyls, sugars and branched sugars.

Terminal modification also provides a useful procedure to conjugate thecytotoxins to the nucleic acid, modify cell type specificity,pharmacokinetics, nuclear permeability, and absolute cell uptake ratefor oligonucleotide pharmaceutical agents. For example, an array ofsubstitutions at the 5′ and 3′ ends to include reactive groups areknown, which allow covalent attachment of the cytotoxins. See, e.g.,OLIGODEOXYNUCLEOTIDES: ANTISENSE INHIBITORS OF GENE EXPRESSION, (1989)Cohen, Ed., CRC Press; PROSPECTS FOR ANTISENSE NUCLEIC ACID THERAPEUTICSFOR CANCER AND AIDS, (1991), Wickstrom, Ed., Wiley-Liss; GENEREGULATION: BIOLOGY OF ANTISENSE RNA AND DNA, (1992) Erickson and Izant,Eds., Raven Press; and ANTISENSE RNA AND DNA, (1992), Murray, Ed.,Wiley-Liss. For general methods relating to antisense compounds, see,ANTISENSE RNA AND DNA, (1988), D. A. Melton, Ed., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

The targeting agent is generally coupled to the cytotoxin via a covalentbond. The covalent bond may be non-reversible, partially reversible, orfully reversible. The degree of reversibility corresponds to thesusceptibility of the targeting agent-cytotoxin complex to in vivodegradation.

In a preferred embodiment, the bond is reversible (e.g., easily cleaved)or partially reversible (e.g., partially or slowly cleaved). Cleavage ofthe bond can occur through biological or physiological processes. Thephysiological/biological processes cleave bonds at any selected locationwithin the complex (e.g., removing an ester group or other protectinggroup that is coupled to an otherwise sensitive chemical functionality)before or after cleaving the bond between the cytotoxin and the linker,resulting in partially degraded complexes. Other cleavages can alsooccur, for example, between the linker and targeting agent.

For rapid degradation of the complex after administration, circulatingenzymes in the plasma (e.g., amidases, reductases) are generally reliedupon to cleave the dendrimer from the pharmaceutical agent. Theseenzymes can include non-specific aminopeptidases and esterases,dipeptidyl carboxy peptidases, proteases of the blood clotting cascade,and the like.

Alternatively, cleavage is through a nonenzymatic process. For example,chemical hydrolysis may be initiated by differences in pH experienced bythe complex following delivery. In such a case, the complex may becharacterized by a high degree of chemical lability at physiological pHof 7.4, while exhibiting higher stability at an acidic or basic pH inthe delivery vehicle. An exemplary complex, which is cleaved in such aprocess is a complex incorporating a N-Mannich base linkage within itsframework.

In most cases, cleavage of the complex will occur during or shortlyafter administration. However, in other embodiments, cleavage does notoccur until the complex reaches the pharmaceutical agent's site ofaction.

The susceptibility of the cytotoxin-targeting agent complexes todegradation can be ascertained through studies of the hydrolytic orenzymatic conversion of the complex to the unbound pharmaceutical agent.Generally, good correlation between in vitro and in vivo activity isfound using this method. See, e.g., Phipps et al., J. Pharm. Sciences78:365 (1989). The rates of conversion are readily determined, forexample, by spectrophotometric methods or by gas-liquid or high pressureliquid chromatography. Half-lives and other kinetic parameters may thenbe calculated using standard techniques. See, e.g., Lowry et al.MECHANISM AND THEORY IN ORGANIC CHEMISTRY, 2nd Ed., Harper & Row,Publishers, New York (1981).

Spacer Groups (“L^(x)”)

In addition to the cleaveable group, one or more linker groups areoptionally introduced between the cytotoxin and the targeting agent.Spacer groups contain at least two reactive functional groups.Typically, one chemical functionality of the spacer group bonds to achemical functionality of the cytotoxin, while the other chemicalfunctionality of the spacer group is used to bond to a chemicalfunctionality of the targeting agent or the cleaveable linker. Examplesof chemical functionalities of spacer groups include hydroxy, mercapto,carbonyl, carboxy, amino, ketone, and mercapto groups. The spacer mayalso be a component of the cleaveable linker, in which case it isgenerally denoted as LX, where “x” is an integer.

The linkers, represented by L^(x) are generally substituted orunsubstituted alkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl or a substituted or unsubstituted heteroalkylgroup.

Exemplary spacer groups include, for example, 6-aminohexanol,6-mercaptohexanol, 10-hydroxydecanoic acid, glycine and other aminoacids, 1,6-hexanediol, P-alanine, 2-aminoethanol, cysteamine(2-aminoethanethiol), 5-aminopentanoic acid, 6-aminohexanoic acid,3-maleimidobenzoic acid, phthalide, α-substituted phthalides, thecarbonyl group, aminal esters, nucleic acids, peptides and the like.

The spacer can serve to introduce additional molecular mass and chemicalfunctionality into the cytotoxin-targeting agent complex. Generally, theadditional mass and functionality will affect the serum half-life andother properties of the complex. Thus, through careful selection ofspacer groups, cytotoxin complexes with a range of serum half-lives canbe produced.

Reactive Functional Groups

For clarity of illustration the succeeding discussion focuses on theconjugation of a cytotoxin of the invention to a targeting agent. Thefocus exemplifies one embodiment of the invention from which, others arereadily inferred by one of skill in the art. No limitation of theinvention is implied, by focusing the discussion on a single embodiment.

Exemplary compounds of the invention bear a reactive functional group,which is generally located on a substituted or unsubstituted alkyl orheteroalkyl chain, allowing their facile attachment to another species.A convenient location for the reactive group is the terminal position ofthe chain.

Reactive groups and classes of reactions useful in practicing thepresent invention are generally those that are well known in the art ofbioconjugate chemistry. Currently favored classes of reactions availablewith reactive cytotoxin analogues are those which proceed underrelatively mild conditions. These include, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982.

Exemplary reaction types include the reaction of carboxyl groups andvarious derivatives thereof including, but not limited to,N-hydroxysuccinimide esters, N-hydroxybenztriazole esters, acid halides,acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl,alkynyl and aromatic esters. Hydroxyl groups can be converted to esters,ethers, aldehydes, etc. Haloalkyl groups are converted to new species byreaction with, for example, an amine, a carboxylate anion, thiol anion,carbanion, or an alkoxide ion. Dienophile (e.g., maleimide) groupsparticipate in Diels-Alder. Aldehyde or ketone groups can be convertedto imines, hydrazones, semicarbazones or oximes, or via such mechanismsas Grignard addition or alkyllithium addition. Sulfonyl halides reactreadily with amines, for example, to form sulfonamides. Amine orsulfhydryl groups are, for example, acylated, alkylated or oxidized.Alkenes, can be converted to an array of new species usingcycloadditions, acylation, Michael addition, etc. Epoxides react readilywith amines and hydroxyl compounds.

One skilled in the art will readily appreciate that many of theselinkages may be produced in a variety of ways and using a variety ofconditions. For the preparation of esters, see, e.g., March supra at1157; for thioesters, see, March, supra at 362-363, 491, 720-722, 829,941, and 1172; for carbonates, see, March, supra at 346-347; forcarbamates, see, March, supra at 1156-57; for amides, see, March supraat 1152; for ureas and thioureas, see, March supra at 1174; for acetalsand ketals, see, Greene et al. supra 178-210 and March supra at 1146;for acyloxyalkyl derivatives, see, PRODRUGS: TOPICAL AND OCULAR DRUGDELIVERY, K. B. Sloan, ed., Marcel Dekker, Inc., New York, 1992; forenol esters, see, March supra at 1160; for N-sulfonylimidates, see,Bundgaard et al., J. Med. Chem., 31:2066 (1988); for anhydrides, see,March supra at 355-56, 636-37, 990-91, and 1154; for N-acylamides, see,March supra at 379; for N-Mannich bases, see, March supra at 800-02, and828; for hydroxymethyl ketone esters, see, Petracek et al. Annals NYAcad. Sci., 507:353-54 (1987); for disulfides, see, March supra at 1160;and for phosphonate esters and phosphonamidates.

The reactive functional groups can be chosen such that they do notparticipate in, or interfere with, the reactions necessary to assemblethe reactive autoinducer analogue. Alternatively, a reactive functionalgroup can be protected from participating in the reaction by thepresence of a protecting group. Those of skill in the art willunderstand how to protect a particular functional group from interferingwith a chosen set of reaction conditions. For examples of usefulprotecting groups, See Greene et al., PROTECTIVE GROUPS IN ORGANICSYNTHESIS, John Wiley & Sons, New York, 1991.

Typically, the targeting agent is linked covalently to a cytotoxin usingstandard chemical techniques through their respective chemicalfunctionalities. Optionally, the dendrimer or agent is coupled to theagent through one or more spacer groups. The spacer groups can beequivalent or different when used in combination.

Generally, prior to forming the linkage between the cytotoxin and thetargeting (or other) agent, and optionally, the spacer group, at leastone of the chemical functionalities will be activated. One skilled inthe art will appreciate that a variety of chemical functionalities,including hydroxy, amino, and carboxy groups, can be activated using avariety of standard methods and conditions. For example, a hydroxylgroup of the cytotoxin or targeting agent can be activated throughtreatment with phosgene to form the corresponding chloroformate, orp-nitrophenylchloroformate to form the corresponding carbonate.

In an exemplary embodiment, the invention makes use of a targeting agentthat includes a carboxyl functionality. Carboxyl groups may be activatedby, for example, conversion to the corresponding acyl halide or activeester. This reaction may be performed under a variety of conditions asillustrated in March, supra pp. 388-89. In an exemplary embodiment, theacyl halide is prepared through the reaction of the carboxyl-containinggroup with oxalyl chloride. The activated agent is reacted with acytotoxin or cytotoxin-linker arm combination to form a conjugate of theinvention. Those of skill in the art will appreciate that the use ofcarboxyl-containing targeting agents is merely illustrative, and thatagents having many other functional groups can be conjugated to thedendrimers of the invention.

When the compound of the invention is conjugated to a detectable label,the label is preferably a member selected from the group consisting ofradioactive isotopes, fluorescent agents, fluorescent agent precursors,chromophores, enzymes and combinations thereof. Methods for conjugatingvarious groups to antibodies are well known in the art. For example, adetectable label that is frequently conjugated to an antibody is anenzyme, such as horseradish peroxidase, alkaline phosphatase,β-galactosidase, and glucose oxidase.

Detectable Labels

The particular label or detectable group used in conjunction with thecompounds and methods of the invention is generally not a criticalaspect of the invention, as long as it does not significantly interferewith the activity or utility of the compound of the invention. Thedetectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and colorimetric labels such ascolloidal gold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.).

The label may be coupled directly or indirectly to a compound of theinvention according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to a component ofthe conjugate. The ligand then binds to another molecules (e.g.,streptavidin) molecule, which is either inherently detectable orcovalently bound to a signal system, such as a detectable enzyme, afluorescent compound, or a chemiluminescent compound.

Components of the conjugates of the invention can also be conjugateddirectly to signal generating compounds, e.g., by conjugation with anenzyme or fluorophore. Enzymes of interest as labels will primarily behydrolases, particularly phosphatases, esterases and glycosidases, oroxidotases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that may be used, see, U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Fluorescent labels are presently preferred as they have the advantage ofrequiring few precautions in handling, and being amenable tohigh-throughput visualization techniques (optical analysis includingdigitization of the image for analysis in an integrated systemcomprising a computer). Preferred labels are typically characterized byone or more of the following: high sensitivity, high stability, lowbackground, low environmental sensitivity and high specificity inlabeling. Many fluorescent labels are commercially available from theSIGMA chemical company (Saint Louis, Mo.), Molecular Probes (Eugene,Oreg.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, Md.),Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), and Applied Biosystems (Foster City, Calif.), as well asmany other commercial sources known to one of skill. Furthermore, thoseof skill in the art will recognize how to select an appropriatefluorophore for a particular application and, if it not readilyavailable commercially, will be able to synthesize the necessaryfluorophore de novo or synthetically modify commercially availablefluorescent compounds to arrive at the desired fluorescent label.

In addition to small molecule fluorophores, naturally occurringfluorescent proteins and engineered analogues of such proteins areuseful in the present invention. Such proteins include, for example,green fluorescent proteins of cnidarians (Ward et al., Photochem.Photobiol. 35:803-808 (1982); Levine et al., Comp. Biochem. Physiol.,72B:77-85 (1982)), yellow fluorescent protein from Vibrio fischeristrain (Baldwin et al., Biochemistry 29:5509-15 (1990)),Peridinin-chlorophyll from the dinoflagellate Symbiodinium sp. (Morriset al., Plant Molecular Biology 24:673:77 (1994)), phycobiliproteinsfrom marine cyanobacteria, such as Synechococcus, e.g., phycoerythrinand phycocyanin (Wilbanks et al., J. Biol. Chem. 268:1226-35 (1993)),and the like.

Pharmaceutical Formulations

In another preferred embodiment, the present invention provides apharmaceutical formulation comprising a compound of the invention and apharmaceutically acceptable carrier.

In a still further preferred embodiment, the invention provides apharmaceutical formulation including a pharmaceutically acceptablecarrier and a conjugate of a targeting agent with a cytotoxin of theinvention.

The compounds described herein, or pharmaceutically acceptable additionsalts or hydrates thereof, can be delivered to a patient using a widevariety of routes or modes of administration. Suitable routes ofadministration include, but are not limited to, inhalation, transdermal,oral, rectal, transmucosal, intestinal and parenteral administration,including intramuscular, subcutaneous and intravenous injections.

As used herein, the terms “administering” or “administration” areintended to encompass all means for directly and indirectly delivering acompound to its intended site of action.

The compounds described herein, or pharmaceutically acceptable saltsand/or hydrates thereof, may be administered singly, in combination withother compounds of the invention, and/or in cocktails combined withother therapeutic agents. Of course, the choice of therapeutic agentsthat can be co-administered with the compounds of the invention willdepend, in part, on the condition being treated.

For example, when administered to patients suffering from a diseasestate caused by an organism that relies on an autoinducer, the compoundsof the invention can be administered in cocktails containing agents usedto treat the pain, infection and other symptoms and side effectscommonly associated with the disease. Such agents include, e.g.,analgesics, antibiotics, etc.

When administered to a patient undergoing cancer treatment, thecompounds may be administered in cocktails containing anti-cancer agentsand/or supplementary potentiating agents. The compounds may also beadministered in cocktails containing agents that treat the side-effectsof radiation therapy, such as anti-emetics, radiation protectants, etc.

Supplementary potentiating agents that can be co-administered with thecompounds of the invention include, e.g., tricyclic anti-depressantdrugs (e.g., imipramine, desipramine, amitriptyline, clomipramine,trimipramine, doxepin, nortriptyline, protriptyline, amoxapine andmaprotiline); non-tricyclic and anti-depressant drugs (e.g., sertraline,trazodone and citalopram); Ca⁺² antagonists (e.g., verapamil,nifedipine, nitrendipine and caroverine); amphotericin; triparanolanalogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine);antihypertensive drugs (e.g., reserpine); thiol depleters (e.g.,buthionine and sulfoximine); and calcium leucovorin.

The active compound(s) of the invention are administered per se or inthe form of a pharmaceutical composition wherein the active compound(s)is in admixture with one or more pharmaceutically acceptable carriers,excipients or diluents. Pharmaceutical compositions for use inaccordance with the present invention are typically formulated in aconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries, which facilitateprocessing of the active compounds into preparations which, can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired. to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations, which can be used orally, include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents may be added, such as thecross-linked polyvinyl pyrrolidone, agar, or alginic acid or a saltthereof such as sodium alginate.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation or transcutaneous delivery (e.g.,subcutaneously or intramuscularly), intramuscular injection or atransdermal patch. Thus, for example, the compounds may be formulatedwith suitable polymeric or hydrophobic materials (e.g., as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Libraries

Also within the scope of the present invention are libraries of thecytotoxin, cytotoxin-linker and agent-linker conjugates of thecytotoxins and linkers of the invention. Exemplary libraries include atleast 10 compounds, more preferably at least 100 compound, even morepreferably at least 1000 compounds and still more preferably at least100,000 compounds. The libraries in a form that is readily queried for aparticular property, e.g., cytotoxicity, cleavage of a linker by anenzyme, or other cleavage reagent. Exemplary forms include chip formats,microarrays, and the like.

Parallel, or combinatorial, synthesis has as its primary objective thegeneration of a library of diverse molecules which all share a commonfeature, referred to throughout this description as a scaffold. Bysubstituting different moieties at each of the variable parts of thescaffold molecule, the amount of space explorable in a library grows.Theories and modern medicinal chemistry advocate the concept of occupiedspace as a key factor in determining the efficacy of a given compoundagainst a given biological target. By creating a diverse library ofmolecules, which explores a large percentage of the targeted space, theodds of developing a highly efficacious lead compound increasedramatically.

Parallel synthesis is generally conducted on a solid phase support, suchas a polymeric resin. The scaffold, or other suitable intermediate iscleavably tethered to the resin by a chemical linker. Reactions arecarried out to modify the scaffold while tethered to the particle.Variations in reagents and/or reaction conditions produce the structuraldiversity, which is the hallmark of each library.

Parallel synthesis of “small” molecules (non-oligomers with a molecularweight of 200-1000) was rarely attempted prior to 1990. See, forexample, Camps. et al., Annaks de Quimica, 70: 848 (1990). Recently,Ellmann disclosed the solid phase-supported parallel (also referred toas “combinatorial”) synthesis of eleven benzodiazepine analogs alongwith some prostaglandins and beta-turn mimetics. These disclosures areexemplified in U.S. Pat. No. 5,288,514. Another relevant disclosure ofparallel synthesis of small molecules may be found in U.S. Pat. No.5,324,483. This patent discloses the parallel synthesis of between 4 and40 compounds in each of sixteen different scaffolds. Chen et al. havealso applied organic synthetic strategies to develop non-peptidelibraries synthesized using multi-step processes on a polymer support.(Chen et al., J. Am. Chem. Soc., 116: 2661-2662 (1994)).

Once a library of unique compounds is prepared, the preparation of alibrary of immunoconjugates, or antibodies can be prepared using thelibrary of autoinducers as a starting point and using the methodsdescribed herein.

Kits

In another aspect, the present invention provides kits containing one ormore of the compounds or compositions of the invention and directionsfor using the compound or composition. In an exemplary embodiment, theinvention provides a kit for conjugating a linker arm of the inventionto another molecule. The kit includes the linker, and directions forattaching the linker to a particular functional group. Other formats forkits will be apparent to those of skill in the art and are within thescope of the present invention.

Methods

In addition to the compositions and constructs described above, thepresent invention also provides a number of methods that can bepracticed utilizing the compounds and conjugates of the invention.

Purification

In another exemplary embodiment, the present invention provides a methodfor isolating a molecular target for a cytotoxin of the invention, whichbinds to a molecule having as a portion of its structure the groupaccording to Formula I. The method preferably comprises, contacting acellular preparation that includes the target with an immobilizedcompound according Formula I, thereby forming a complex between thereceptor and the immobilized compound.

The cytotoxin of the invention can be immobilized on an affinity supportby any art-recognized means. Alternatively, the cytotoxin can beimmobilized using one or more of the linkers of the invention.

In yet another exemplary embodiment, the invention provides an affinitypurification matrix that includes a linker of the invention.

The method of the invention for isolating a target will typicallyutilize one or more affinity chromatography techniques. Affinitychromatography enables the efficient isolation of species such asbiological molecules or biopolymers by utilizing their recognition sitesfor certain supported chemical structures with a high degree ofselectivity. The literature is replete with articles, monographs, andbooks on the subject of affinity chromatography, including such topicsas affinity chromatography supports, crosslinking members, ligands andtheir preparation and use. A sampling of those references includes:Ostrove, Methods Enzymol. 182: 357-71 (1990); Ferment, Bioeng. 70:199-209 (1990). Huang et al., J. Chromatogr. 492: 431-69 (1989);“Purification of enzymes by heparin-Sepharose affinity chromatography,”J. Chromatogr., 184: 335-45 (1980); Farooqi, Enzyme Eng., 4: 441-2(1978); Nishikawa, Chem. Technol., 5(9): 564-71 (1975); Guilford et al.,in, PRACT. HIGH PERFORM. LIQ. CHROMATOGR., Simpson (ed.), 193-206(1976); Nishikawa, Proc. Int. Workshop Technol. Protein Sep. Improv.Blood Plasma Fractionation, Sandberg (ed.), 422-35; (1977) “Affinitychromatography of enzymes,” Affinity Chromatogr., Proc. Int. Symp.25-38, (1977) (Pub. 1978); and AFFINITY CHROMATOGRAPHY: A PRACTICALAPPROACH, Dean et al. (ed.), IRL Press Limited, Oxford, England (1985).Those of skill in the art have ample guidance in developing particularaffinity chromatographic methods utilizing the materials of theinvention.

In the present method, affinity chromatographic media of varyingchemical structures can be used as supports. For example, agarose gelsand cross-linked agarose gels are useful as support materials, becausetheir hydrophilicity makes them relatively free of nonspecific binding.Other useful supports include, for example, controlled-pore glass (CPG)beads, cellulose particles, polyacrylamide gel beads and Sephadex™ gelbeads made from dextran and epichlorohydrin.

Treatment of Disease

The cytotoxins of the invention are active, potent duocarmycinderivatives. The parent agents are exceptionally potent antitumorantibiotics that derive their biological effects through the reversible,stereoelectronically controlled sequence selective alkylation of DNA(Boger et al. J. Org. Chem. 55: 4499 (1990); Boger et al. J. Am. Chem.Soc. 112: 8961 (1990); Boger et al., J. Am. Chem. Soc. 113: 6645 (1991);Boger et al. J. Am. Chem. Soc. 115: 9872 (1993); Boger et al., Bioorg.Med. Chem. Lett. 2: 759 (1992)). Subsequent to the initial disclosure ofthe duocarmycins, extensive efforts have been devoted to elucidating theDNA alkylation selectivity of the duocarmycins and its structuralorigin.

In yet a further embodiment, the present invention provides a method ofkilling a cell. The method includes administering to the cell an amountof a compound of the invention sufficient to kill said cell. In anexemplary embodiment, the compound is administered to a subject bearingthe cell. In a further exemplary embodiment, the administration servesto retard of stop the growth of a tumor that includes the cell.

Effective Dosages

Pharmaceutical compositions suitable for use with the present inventioninclude compositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., in an amount effective toachieve its intended purpose. The actual amount effective for aparticular application will depend, inter alia, on the condition beingtreated. For example, when administered in methods to reduce sickle celldehydration and/or delay the occurrence of erythrocyte sickling ordistortion in situ, such compositions will contain an amount of activeingredient effective to achieve this result. Determination of aneffective amount is well within the capabilities of those skilled in theart, especially in light of the detailed disclosure herein.

For any compound described herein, the therapeutically effective amountcan be initially determined from cell culture assays. Target plasmaconcentrations will be those concentrations of active compound(s) thatare capable of inhibition cell growth or division. In preferredembodiments, the cellular activity is at least 25% inhibited. Targetplasma concentrations of active compound(s) that are capable of inducingat least about 50%, 75%, or even 90% or higher inhibition of cellularactivity are presently preferred. The percentage of inhibition ofcellular activity in the patient can be monitored to assess theappropriateness of the plasma drug concentration achieved, and thedosage can be adjusted upwards or downwards to achieve the desiredpercentage of inhibition.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a circulating concentration thathas been found to be effective in animals. The dosage in humans can beadjusted by monitoring cellular inhibition and adjusting the dosageupwards or downwards, as described above.

A therapeutically effective dose can also be determined from human datafor compounds which are known to exhibit similar pharmacologicalactivities. The applied dose can be adjusted based on the relativebioavailability and potency of the administered compound as comparedwith the known compound.

Adjusting the dose to achieve maximal efficacy in humans based on themethods described above and other methods as are well-known in the artis well within the capabilities of the ordinarily skilled artisan.

In the case of local administration, the systemic circulatingconcentration of administered compound will not be of particularimportance. In such instances, the compound is administered so as toachieve a concentration at the local area effective to achieve theintended result.

For use in the prophylaxis and/or treatment of diseases related toabnormal cellular proliferation, a circulating concentration ofadministered compound of about 0.001 μM to 20 μM is preferred, withabout 0.01 μM to 5 μM being preferred.

Patient doses for oral administration of the compounds described herein,typically range from about 1 mg/day to about 10,000 mg/day, moretypically from about 10 mg/day to about 1,000 mg/day, and most typicallyfrom about 50 mg/day to about 500 mg/day. Stated in terms of patientbody weight, typical dosages range from about 0.01 to about 150mg/kg/day, more typically from about 0.1 to about 15 mg/kg/day, and mosttypically from about 1 to about 10 mg/kg/day.

For other modes of administration, dosage amount and interval can beadjusted individually to provide plasma levels of the administeredcompound effective for the particular clinical indication being treated.For example, in one embodiment, a compound according to the inventioncan be administered in relatively high concentrations multiple times perday. Alternatively, it may be more desirable to administer a compound ofthe invention at minimal effective concentrations and to use a lessfrequent administration regimen. This will provide a therapeutic regimenthat is commensurate with the severity of the individual's disease.

Utilizing the teachings provided herein, an effective therapeutictreatment regimen can be planned which does not cause substantialtoxicity and yet is entirely effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration and the toxicity profile of the selected agent.

The compounds, compositions and methods of the present invention arefurther illustrated by the examples that follow. These examples areoffered to illustrate, but not to limit the claimed invention.

EXAMPLES Example 1 1.1 Material and Methods

In the examples below, unless otherwise stated, temperatures are givenin degrees Celsius (° C.); operations were carried out at room orambient temperature (typically a range of from about 18-25° C.;evaporation of solvent was carried out using a rotary evaporator underreduced pressure (typically, 4.5-30 mmHg) with a bath temperature of upto 60° C.; the course of reactions was typically followed by TLC andreaction times are provided for illustration only; melting points areuncorrected; products exhibited satisfactory ¹H-NMR and/ormicroanalytical data; yields are provided for illustration only; and thefollowing conventional abbreviations are also used: mp (melting point),L (liter(s)), mL (milliliters), mmol (millimoles), g (grams), mg(milligrams), min (minutes), LC-MS (liquid chromatography-massspectrometry) and h (hours).

¹H-NMR spectra were measured on a Varian Mercury 300 MHz spectrometerand were consistent with the assigned structures. Chemical shifts werereported in parts per million (ppm) downfield from tetramethylsilane.Electrospray mass spectra were recorded on a Perkin Elmer Sciex API 365mass spectrometer. Elemental analyses were performed by RobertsonMicrolit Laboratories, Madison, N.J. Silica gel for flash chromatographywas E. Merck grade (230-400 mesh). Reverse-Phase analytical HPLC wasperformed on either a HP 1100 or a Varian ProStar 210 instrument with aPhenomenex Luna 5 μm C-18(2) 150 mm×4.6 mm column or a VarianMicrosorb-MV 0.1 μm C-18 150 mm×4.6 mm column. A flow rate of 1 mL/minwas with either a gradient of 0% to 50% buffer B over 15 minutes or 10%to 100% buffer B over 10 minutes with detection by UV at 254 nm. BufferA, 20 mM ammonium formate+20% acetonitrile or 0.1% trifluoroacetic acidin acetonitrile; buffer B, 20 mM ammonium formate+80% acetonitrile or0.1% aqueous trifluoroacetic acid. Reverse phase preparative HPLC wereperformed on a Varian ProStar 215 instrument with a Waters Delta Pak 15μm C-18 300 mm×7.8 mm column.

1.2 Synthetic Methodology

1.2a Synthesis of Compound 134

The compounds of Formula I are readily prepared by reacting theappropriate spirocyclopropylcyclohexadienyl analog (Compounds B) withthe activated heterocyclic compounds A using sodium hydride inN,B-dimethylformamide (DMF) or tetrahydrofuran (THF). The resultingcompound 28 is then converted to compound 29 by treatment with theappropriate halo-acid, such as hydrochloric acid. Compound 29 is reducedby catalytic hydrogenation to give compound 65, which is coupled anactivated ester to give compound 134, a compound of Formula I.

Other compounds of Formula I are prepared according to publishedprocedures, which are modified to make additional analogs usingprocedures well known to those skilled in the art, such as reductions,oxidations, additions, aqueous extractions, evaporation, andpurification.

1.2b Synthesis of Compound A

To a solution of 5-nitro-2-carboxylic acid (0.83 g, 4.0 mmol) inN,N-dimethylformamide (60 mL) at 0° C. was added EDC (1.15 g, 6.0 mmol).The resulting suspension was stirred at 0° C. for 45 min, by which timethe EDC had completely dissolved. 4-Nitrophenol (0.83 g, 6.0 mmol) andDMAP (0.73 g, 6.0 mmol) were added and the resulting mixture stirred atambient temperature. After 13 hours, the mixture was diluted with ethylacetate and washed with a 10% aqueous citric acid solution twice,followed by water, and brine, then dried over Na₂SO₄, filtered andconcentrated in vacuo. Purification of the resultant residue by flashcolumn chromatography on silica gel (7% ethyl acetate in methylenechloride) afforded 1.02 g (78%) of A as a yellow solid: ¹H NMR (CDCl₃) δ9.0 (br s, 1H), 8.2 (d, 2H), 7.8 (m, 2H), 7.4 (d, 2H), 7.3 (s, 1H), 6.8(s, 1H).

1.2c Synthesis of Compound 28

To a solution of B (20 mg, 0.08 mmol) in N,N-dimethylformamide (1.0 mL)at −40° C. was added a suspension of sodium hydride (4.0 mg, 0.1 mmol,60% in oil) in N,N-dimethylformamide (1.0 mL). The resulting mixture wasallowed to warm to 0° C. slowly (1.5 h), then cooled back to −40° C. A(37 mg, 0.1 mmol) was added and the mixture allowed to warm to 0° C.slowly (1.5 h) where it was kept for 20 min. The mixture was cooled to−30° C., quenched with acetic acid (10 μL), stirred for 10 min, dilutedwith ethyl acetate, then washed with water then brine. The organic layerwas separated and dried over MgSO₄, filtered and concentrated in vacuo.Purification by flash column chromatography on silica gel (50% to 100%ethyl acetate in methylene chloride) afforded 16.3 mg (43%) of 28 as aslightly yellow solid: ¹H NMR (CDCl₃) δ 11.3 (br s, 1H), 9.4 (br s, 1H),7.4 (m, 2H), 7.1 (s, 1H), 6.95 (s, 1H), 6.8 (s, 1H), 4.4 (s, 2H), 3.8(s, 3H), 3.8 (m, 1H), 2.6 (s, 3H), 2.4 (dd, 1H), 1.4 (m, 1H). ESMS m/z490 (M-H)⁻.

1.2d Synthesis of Compound 29

To a solution of 28 (50 mg, 0.103 mmol) in N,N-dimethylformamide (1.0mL) was treated with 1 mL of anhydrous hydrochloric acid (1.0 M indioxane). The resulting solution was stirred at ambient temperature for30 min, then concentrated of solvent. Purification of the resultingresidue by flash column chromatography on silica gel (50% to 100% ethylacetate in methylene chloride) afforded 50 mg (100%) of 29 as a slightlyyellow solid: ¹H NMR (CDCl₃) δ 11.3 (br s, 1H), 9.4 (br s, 1H), 7.4 (m,2H), 7.1 (s, 1H), 6.95 (s, 1H), 6.8 (s, 1H), 4.4 (s, 2H), 3.8 (s, 3H),3.8 (m, 1H), 2.6 (s, 3H), 2.4 (dd, 1H), 1.4 (m, 1H).

1.2d Synthesis of Compound 65

To a solution of 29 (110 mg, 0.184 mmol) in 1:1 methanol:methylenechloride (20 ml) was added 10% palladium on carbon (100 mg). The mixturewas hydrogenated on a Parr apparatus at 50 psi for one hour. The mixturewas filtered over Celite, rinsed with methylene chloride, thenconcentrated in vacuo to give 94 mg (90% yield) of 65 as a yellow solid:¹H NMR (CDCl₃) δ 7.8-7.3 (m, 5H), 4.4 (m, 3H), 3.8 (m, 5H), 3.4 (s, 3H).ESMS m/z 454 (M-H)⁻.

1.2e Synthesis of Compound 134

Compound C (19 mg, 0.044 mmol) and HATU (50 mg, 0.132 mmol) weredissolved in dimethylformamide (2 ml)and the N-methylmorpholine (19.3μl, 0.176 mmol) added. After 15 minutes, a solution of 65 (20 mg, 0.044mmol) in dimethylformamide (1 ml) was added. The reaction mixture wasstirred for 16 hours at room temperature, then concentrated in vacuo.The resulting solid was rinsed with water and saturated aqueous sodiumbicarbonate, dried over vacuum, then washed with ethyl acetate. Thecrude product was purified by flash chromatography using silica gel and5% methanol/methylene chloride to give 15 mg (39% yield) of 134: ¹H NMR(DMSO): δ 11.4 (br s, 1H), 11.0 (br s, 1H), 10.9 (s, 1H), 8.5 (s, 1H),8.3 (br s, 2H), 8.0 (m, 2H), 7.9 (s, 1H), 7.7 (m, 3H), 7.5 (d, 1H), 7.3(t, 2H), 4.6 (m, 2H), 4.5 (d, 2H), 4.3 (m, 1H), 3.8 (s, 3H), 3.7 (d,1H), 3.5 (t, 1H), 2.7 (s, 3H), 1.7 (m, 2H), 0.9 (d, 6H).

In a similar manner the following compounds were prepared:

46: ¹H NMR (CDCl₃): δ 8.9 (s, 1H), 8.7 (s, 1H), 8.4 (dd, 1H), 8.1 (br s,1H), 7.75 (d, 1H), 7.65 (s, 1H), 4.8 (d, 1H), 4.55 (m, 2H), 3.9 (s, 3H),3.85 (s, 1H), 3.7 (m, 4H), 3.4 (t, 1H), 2.7 (s, 3H), 2.5 (br s, 4H), 2.4(s, 3H).

95: ¹H NMR (DMSO): δ 12 (s, 1H), 10.6 (d, 1H), 8.25 (s, 1H), 8.2 (br s,1H), 8.1 (s, 1H), 7.7 (m, 5H), 7.5 (d, 1H), 4.6 (t, 1H), 4.5 (d, 1H),4.4 (m, 2H), 4.1 (m, 1H), 3.9 (d, 2H), 3.8 (s, 3H), 3.3 (m, 10H), 2.8(s, 3H), 2.6 (s, 3H), 1.6 (br s, 3H), 0.9 (s, 6H).

47: ¹H NMR (CDCl₃): δ 9.1 (br s, 1H), 8.1 (br s, 1H), 7.4 (t, 2H), 6.9(s, 1H), 6.8 (dd, 1H), 4.8 (d, 1H), 4.5 (m, 2H), 3.9 (s, 3H), 3.85 (m,3H), 3.7 (m, 2H), 3.4 (t, 1H), 2.7 (s, 3H), 2.6 (br s, 4H), 2.4 (s, 3H).

52: ¹H NMR (DMSO): δ 12.5 (s, 1H), 11.8 (s, 1H), 10.4 (s, 1H), 8.4 (s,1H), 7.8 (m, 5H), 7.5 (m, 2H), 7.3 (t, 1H), 7.1 (t, 1H), 6.7 (s, 1H),4.6 (m, 4H), 3.8 (s, 3H), 2.5 (s, 3H).

108: ¹H NMR (DMSO): δ 10.9 (s, 1H), 10.7 (s, 1H), 10.0 (s, 1H), 8.5 (s,1H), 8.3 (s, 1H), 8.1 (m, 5H), 7.8 (m, 5H), 7.5 (m, 2H), 7.3 (m, 5H),7.1 (m, 5H), 5.0 (m, 2H), 4.8 (m, 1H), 4.6 (m, 2H), 4.3 (m, 2H), 4.1 (t,2H), 3.9 (m, 1H), 3.7 (m, 4H), 3.0 (m, 6H), 2.6 (s, 2H), 2.3 (t, 1H),1.8 (s, 3H), 1.5 (m, 9H), 1.3 (m, 4H), 0.8 (m, 6H).

43: ¹H NMR (DMSO): δ 12.1 (s, 2H), 11.8 (s, 1H), 10.5 (d, 1H), 8.3 (s,1H), 8.0 (s, 1H), 7.8 (m, 5H), 7.6 (s, 1H), 7.2 (d, 2H), 4.7 (m, 2H),4.5 (m, 3H), 3.8 (s, 3H), 3.5 (m, 2H), 3.2 (m, 2H), 2.9 (s, 3H), 2.7 (s,3H), 2.3 (s, 4H).

153: ¹H NMR (DMSO): δ 12.3 (br s, 1H), 11.7 (br s, 1H), 10.5 (br s, 1H),10.0 (br s, 1H), 8.3 (m, 2H), 7.9 (m, 4H), 7.5 (s, 1H), 7.4 (d, 1H), 7.0(m, 1H), 4.5 (m, 5H), 4.1 (m, 1H), 3.9 (d, 1H), 3.8 (s, 3H), 3.4 (m,8H), 2.9 (br s, 3H), 2.8 (s, 3H), 2.7 (s, 3H), 1.8 (s, 2H), 1.6 (br s,4H), 1.4 (m, 2H), 1.2 (d, 4H), 0.9 (m, 16H).

45: ¹H NMR (DMSO): δ 12.0 (s, 1H), 11.6 (s, 1H), 10.8 (s, 1H), 8.4 (s,1H), 8.3 (d, 2H), 8.0 (s, 1H), 7.8 (m, 3H), 7.6 (s, 1H), 7.4 (t, 1H),4.6 (m, 5H), 3.8 (s, 3H), 3.4 (m, 8H), 2.9 (s, 3H), 2.7 (s, 3H).

115: ¹H NMR (CDCl₃): δ 9.1 (br s, 1H), 8.4 (s, 1H), 8.3 (s, 1H), 7.7 (d,1H), 7.5 (m, 3H), 7.2 (m, 3H), 6.9 (s, 2H), 4.7 (d, 1H), 4.5 (m, 4H),3.9 (s, 3H), 3.5 (m, 14H), 2.6 (s, 3H), 1.3 (t, 3H).

109: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.5 (s, 1H), 10.2 (d, 1H), 8.2 (s,1H), 7.7 (m, 6H), 7.2 (d, 1H), 7.1 (t, 1H), 6.8 (d, 1H), 4.6 (m, 1H),4.4 (d, 2H), 4.3 (m, 2H), 3.7 (s, 3H), 2.6 (s, 3H).

135: ¹H NMR (DMSO): δ 11.4 (br s, 1H), 11.0 (br s, 1H), 10.9 (s, 1H),8.5 (s, 1H), 8.3 (br s, 2H), 8.0 (m, 2H), 7.9 (s, 1H), 7.7 (m, 3H), 7.5(d, 1H), 7.3 (t, 2H), 4.6 (m, 2H), 4.5 (d, 2H), 4.3 (m, 1H), 3.8 (s,3H), 3.7 (d, 1H), 3.5 (t, 1H), 2.7 (s, 3H), 1.7 (m, 2H), 0.9 (d, 6H).

24: ¹H NMR (DMSO): δ 10.8 (s, 1H), 8.6 (s, 1H), 8.3 (m, 5H), 7.9 (s,1H), 7.8 (d, 2H), 7.7 (d, 1H), 7.65 (s, 1H), 7.6 (d, 2H), 7.4 (m, 5H),5.3 (s, 2H), 4.9 (t, 1H), 4.7 (d, 1H), 4.4 (m, 1H), 4.0 (m, 2H).

ESMS m/z 696 (M-H)⁻.

25: ¹H NMR (DMSO): δ 8.6 (s, 1H), 8.3 (m, 5H), 7.8 (m, 3H), 7.6 (m, 3H),7.4 (m, 1H), 4.8 (m, 1H). 4.6 (m, 1H), 4.3 (m, 1H), 4.1 (m, 2H).

ESMS m/z 605 (M-H)⁻.

27: ¹H NMR (DMSO): δ 10.9 (s, 1H), 10.3 (s, 1H), 8.6 (s, 1H), 8.3 (s,1H), 8.2 (d, 1H), 8.1 (m, 2H), 7.8 (m, 3H), 7.6 (d, 1H), 7.3 (s, 1H),6.9 (d, 1H), 6.8 (t, 1H), 6.4 (d, 1H), 4.8 (t, 1H), 4.6 (d, 1H), 4.3 (m,1H), 4.0 (m, 2H).

154: ¹H NMR (DMSO): δ 10.7 (s, 1H), 10.0 (s, 1H), 8.6 (s, 1H), 8.4 (s,1H), 8.2 (s, 1H), 8.1 (m, 3H), 7.9 (m, 5H), 7.7 (m, 1H), 7.6 (m, 3H),7.3 (m, 5H), 5.0 (s, 2H), 4.8 (t, 1H), 4.6 (d, 1H), 4.3 (m, 3H), 4.1 (d,1H), 3.9 (m, 1H), 3.1 (s, 1H), 3.0 (m, 1H), 2.7 (s, 1H), 2.3 (m, 5H),1.6 (m, 5H), 1.4 (t, 2H), 1.2 (d, 3H), 0.9 (m, 12H).

ESMS m/z 1134 (M-H)⁻.

162: ¹H NMR (DMSO): δ 11.6 (s, 1H), 10.9 (s, 1H), 10.5 (s, 1H), 9.9 (s,1H), 8.6 (s, 1H), 8.3 (s, 1H), 8.2 (d, 1H), 8.0 (m, 5H), 7.8 (m, 4H),7.6 (d, 1H), 7.5 (m, 3H), 7.1 (t, 1H), 4.8 (t, 1H), 4.6 (d, 2H), 4.3 (m,3H), 4.1 (d, 1H), 3.9 (m, 1H), 2.4 (m, 2H), 2.3 (m, 3H) 1.5 (m, 9H), 1.2(m, 3H), 0.9 (m, 12H).

ESMS m/z 1044 (M-H)⁻.

79: ¹H NMR (CDCl₃): δ 9.4 (s, 1H), 8.5 (s, 1H), 8.1 (s, 2H), 8.0 (br s,1H), 7.9 (d, 2H), 7.6 (s, 2H), 7.5 (s, 1H), 7.0 (d, 2H), 6.9 (d, 3H),4.7 (d, 1H), 4.5 (m, 2H), 4.2 (m, 1H), 3.95 (s, 3H), 3.85 (s, 3H), 3.7(m, 4H), 3.4 (m, 1H), 2.7 (s, 3H), 2.5 (s, 3H), 2.4 (t, 2H), 1.1 (s,9H), 0.4 (br s, 6H).

80: ¹H NMR (CDCl₃): δ 9.3 (br s, 1H), 8.3 (s, 1H), 8.2 (br s, 1H), 8.0(m, 3H), 7.5 (m, 4H), 7.4 (m, 2H), 7.0 (m, 4H), 4.7 (d, 1H), 4.6 (m,1H), 4.4 (m, 1H), 4.2 (m, 5H), 3.9 (s, 3H), 3.4 (m, 1H), 2.7 (s, 3H),2.9 (s, 3H), 2.3 (m, 2H), 1.1 (s, 9H), 0.4 (br s, 6H).

81: ¹H NMR (CDCl₃): δ 10.5 (s, 1H), 8.8 (s, 1H), 8.6 (d, 2H), 8.0 (d,2H), 7.8 (d, 2H), 7.4 (m, 3H), 7.3 (d, 2H), 7.0 (m, 2H), 6.9 (d, 2H),4.6 (m, 3H), 4.4 (m, 2H), 3.9 (m, 4H), 3.4 (m, 1H), 2.7 (s, 3H), 2.5 (s,3H), 1.0 (s, 9H), 0.3 (s, 6H).

82: ¹H NMR (CDCl₃): δ 8.5 (s, 2H), 8.4 (s, 1H), 8.2 (s, 1H), 8.0 (m,4H), 7.6 (m, 4H), 7.5 (s, 1H), 7.3 (s, 1H), 7.1 (s, 2H), 4.7 (m, 3H),4.55 (m, 1H), 4.45 (m, 1H), 3.9 (m, 4H), 3.4 (m, 1H), 2.7 (s, 3H), 2.5(s, 3H), 1.0 (s, 9H), 0.4 (br s, 6H).

83: ¹H NMR (CDCl₃): δ 9.6 (s, 1H), 8.4 (s, 1H), 8.1 (s, 2H), 8.0 (m,1H), 7.8 (s, 1H), 7.6 (m, 2H), 7.5 (s, 1H), 7.35 (q, 2H), 7.0 (s, 1H),4.7 (d, 1H), 4.55 (m, 1H), 4.45 (m, 1H), 3.9 (m, 4H), 3.4 (m, 1H), 2.7(s, 3H), 1.0 (s, 9H), 0.4 (br s, 6H).

89: ¹H NMR (CDCl₃): δ 9.3 (br s, 1H), 8.4 (br s, 1H), 8.2 (s, 1H), 8.0(br s, 2H), 7.5 (m, 9H), 7.2 (s, 1H), 7.1 (d, 1H), 6.9 (s, 1H), 5.1 (s,2H), 4.7 (d, 1H), 4.55 (m, 1H), 4.45 (m, 1H), 3.9 (m, 4H), 3.4 (m, 1H),2.7 (s, 3H), 1.05 (s, 9H), 0.4 (br s, 6H).

90: ¹H NMR (CDCl₃): δ 9.6 (br s, 1H), 8.5 (br s, 1H), 8.2 (s, 1H), 8.0(m, 2H), 7.5 (m, 8H), 7.3 (m, 2H), 7.1 (d, 1H), 6.6 (d, 1H), 5.2 (s,2H), 4.7 (d, 1H), 4.55 (m, 1H), 4.45 (m, 1H), 3.9 (m, 4H), 3.4 (m, 1H),2.7 (s, 3H), 1.05 (s, 9H), 0.3 (br s, 6H).

163: ¹H NMR (DMSO): δ 12.0 (s, 1H), 11.6 (s, 1H), 10.4 (s, 1H), 10.2 (s,1H), 8.4 (s, 1H), 8.1 (m, 4H), 7.9 (m, 3H), 7.5 (m, 3H), 7.2 (d, 2H),4.9 (s, 2H), 4.7 (m, 1H), 4.6 (m, 1H), 4.5 (m, 1H), 3.9 (m, 4H), 3.6 (m,1H), 2.7 (s, 3H), 2.5 (s, 3H), 1.1 (s, 9H), 0.7 (s, 6H).

92: ¹H NMR (DMSO): δ 12.0 (s, 1H), 10.4 (s, 1H), 10.2 (s, 1H), 8.3 (s,1H), 7.9 (s, 1H), 7.8 (m, 4H), 7.5 (d, 2H), 7.25 (s, 1H), 7.15 (s, 1H),6.9 (m, 3H), 4.7 (m, 3H), 4.6 (m, 1H), 4.5 (m, 1H), 3.9 (m, 2H), 3.8 (m,4H), 3.5 (m, 1H), 3.2 (m, 2H), 2.9 (s, 3H), 2.7 (s, 3H), 2.6 (s, 3H).

98: ¹H NMR (CDCl₃): δ 8.3 (br s, 1H), 8.1 (d, 1H), 8.0 (br s, 1H), 7.95(s, 1H), 7.85 (d, 2H), 7.65 (s, 1H), 7.6 (d, 2H), 7.5 (s, 1H), 7.4 (m,2H), 7.0 (s, 1H), 6.85 (d, 2H), 4.8 (m, 3H), 4.55 (m, 1H), 4.45 (m, 1H),3.9 (m, 6H), 3.4 (m, 1H), 2.7 (s, 3H), 2.5 (s, 3H), 2.4 (m, 2H), 1.05(s, 9H), 0.4 (br s, 6H).

110: ¹H NMR (C₃D₆O): δ 11.3 (br s, 1H), 9.7 (s, 1H), 9.6 (d, 1H), 8.2(s, 1H), 7.9 (m, 2H), 7.7 (d, 2H), 7.6 (m, 3H), 7.5 (d, 1H), 7.2 (m,3H), 6.9 (m, 3H), 6.8 (s, 1H), 4.9 (m, 2H), 4.7 (m, 1H), 4.6 (m, 1H),4.5 (m, 1H), 3.8 (m, 25H), 3.5 (m, 1H), 3.2 (m, 2H), 2.7 (s, 3H), 2.3(m, 4H), 2.0 (s, 3H).

113: ¹H NMR (C₃D₆O): δ 11.3 (br s, 1H), 10.1 (s, 1H), 9.9 (s, 1H), 8.4(s, 1H), 7.9 (m, 3H), 7.7 (m, 2H), 7.5 (m, 3H), 7.4 (m, 1H), 7.2 (s,1H), 7.1 (d, 2H), 6.9 (t, 1H), 5.0 (s, 2H), 4.6 (d, 1H), 4.5 (m, 1H),4.4 (m, 1H), 3.9 (d, 1H), 3.7 (s, 3H), 3.4 (m, 1H), 2.6 (s, 3H), 2.4 (s,3H).

114: ¹H NMR (DMSO): δ 11.9 (br s, 1H), 11.7 (s, 1H), 10.4 (d, 1H), 10.2(t, 1H), 8.3 (s, 1H), 7.9 (s, 1H), 7.8 (m, 7H), 7.5 (m, 2H), 7.0 (m,6H), 4.9 (s, 2H), 4.6 (m, 1H), 4.5 (m, 1H), 4.4 (m, 1H), 3.9 (d, 1H),3.8 (s, 3H), 3.7 (m, 2H), 3.5 (m, 25H), 2.9 (s, 3H), 2.7 (s, 3H), 2.2(m, 2H).

159: ¹H NMR (DMSO): δ 12.0 (m, 1H), 11.9 (br s, 1H), 8.3 (m, 2H), 8.0(m, 4H), 7.6 (m, 2H), 7.3 (s, 1H), 7.1 (d, 1H), 6.9 (s, 1H), 4.5 (m,3H), 4.3 (m, 2H), 3.9 (d, 1H), 3.8 (s, 6H), 3.5 (m, 8H), 3.2 (m, 4H),2.7 (s, 3H), 2.3 (m, 4H), 1.4 (m, 9H), 1.1 (m, 3H), 0.8 (m, 12H).

131: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.2 (s, 1H), 7.7 (m, 1H), 7.5 (s,1H), 7.0 (m, 3H), 6.7 (d, 1H), 4.7 (m, 1H), 4.4 (d, 1H), 4.3 (m, 2H),3.9 (d, 1H), 3.8 (s, 3H), 2.6 (s, 3H).

129: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.2 (s, 1H), 7.8 (m, 4H), 4.5 (m,2H), 4.3 (m, 1H), 3.9 (s, 3H), 3.8 (d, 1H), 3.7 (s, 3H), 3.4 (t, 1H),2.5 (s, 3H).

136: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.2 (s, 1H), 7.65 (d, 2H), 7.55 (s,1H), 7.3 (d, 1H), 7.1 (dd, 1H), 4.6 (m, 1H), 4.5 (m, 1H), 4.3 (m, 1H),3.85 (m, 1H), 3.8 (s, 3H), 3.75 (s, 3H), 3.5 (m, 1H), 2.6 (s, 3H).

137: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.2 (s, 1H), 8.0 (m, 1H), 7.5 (m,3H), 7.2 (s, 1H), 7.0 (m, 1H), 4.6 (m, 1H), 4.5 (m, 1H), 4.4 (m, 1H),3.9 (m, 2H), 3.8 (d, 1H), 3.7 (s, 3H), 3.4 (m, 1H), 2.6 (s, 3H), 2.4 (m,2H), 1.3 (s, 9H).

143: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.2 (s, 1H), 8.0 (m, 2H), 7.7 (m,3H), 7.3 (s, 1H), 7.1 (d, 1H), 4.6 (m, 1H), 4.5 (m, 1H), 4.3 (m, 1H),4.2 (m, 2H), 3.9 (d, 1H), 3.8 (s, 3H), 3.4 (m, 1H), 3.3 (m, 2H), 2.6 (s,3H).

148: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.1 (s, 1H), 7.7 (d, 2H), 7.5 (s,1H), 7.3 (s, 1H), 7.1 (d, 1H), 4.5 (m, 1H), 4.4 (d, 1H), 4.3 (m, 3H),3.8 (m, 1H), 3.7 (s, 3H), 3.5 (m, 3H), 2.8 (s, 6H), 2.5 (s, 3H).

150: ¹H NMR (DMSO): δ 10.3 (s, 1H), 9.4 (s, 1H), 7.8 (m, 1H), 7.5 (m,2H), 7.1 (s, 1H), 6.9 (dd, 1H), 4.5 (m, 4H), 4.3 (m, 1H), 3.8 (s, 3H),3.75 (d, 1H), 3.5 (m, 1H), 3.1 (m, 2H), 2.75 (s, 6H), 2.65 (s, 3H), 2.1(m, 2H).

151: ¹H NMR (DMSO): δ 11.9 (s, 1H), 10.2 (s, 1H), 7.7 (d, 2H), 7.65 (s,1H), 7.55 (s, 1H), 7.25 (d, 1H), 4.6 (m, 1H), 4.5 (m, 1H), 4.4 (m, 1H),3.9 (d, 1H), 3.8 (s, 3H), 3.5 (m, 1H), 3.3 (br m, 8H), 2.9 (s, 3H), 2.6(s, 3H).

251: ¹H NMR (CDCl₃): δ 12.0 (s, 1H), 7.9 (m, 1H), 7.7 (d, 1H), 7.6 (s,1H), 7.4 (s, 1H), 7.2 (dd, 1H), 4.6 (m, 3H), 4.4 (m, 2H), 3.9 (m, 1H),3.8 (s, 3H), 3.7 (m, 1H), 3.6 (m, 2H), 3.4 (m, 4H), 3.2 (m, 2H), 3.0 (m,4H), 2.9 (s, 6H), 2.7 (m, 4H), 2.6 (s, 2H), 1.6 (m, 2H), 1.3 (m, 8H),0.9 (q, 2H).

227: ¹H NMR (CDCl₃): δ 10.3 (s, 1H), 8.7 (s, 1H), 7.7 (d, 2H), 7.5 (m,3H), 7.1 (m, 2H), 6.9 (d, 2H), 4.6 (m, 5H), 4.25 (m, 2H), 4.15 (m, 2H),3.9 (s, 3H), 3.4 (m, 1H), 3.1 (s, 2H), 2.9 (m, 12H), 2.7 (s, 3H), 2.5(s, 6H), 2.3 (s, 3H).

230: ¹H NMR (CDCl₃): δ 10.3 (s, 1H), 8.6 (s, 1H), 7.7 (d, 2H), 7.5 (m,3H), 7.1 (m, 2H), 6.9 (d, 2H), 6.7 (s, 2H), 4.7 (m, 4H), 4.1 (m, 4H),3.9 (s, 3H), 3.5 (m, 2H), 3.4 (m, 1H), 3.2 (m, 4H), 3.1 (s, 2H), 2.8 (m,5H), 2.4 (m, 10H), 2.2 (m, 7H).

166: ESMS m/z 532 (M-H)⁻.

165: ESMS m/z 920 (M-H)⁻.

9: ESMS m/z 966 (M-H)⁻.

17: ESMS m/z 753 (M-H)⁻.

19: ESMS m/z 696 (M-H)⁻.

50: ESMS m/z 800 (M-H)⁻.

174: ¹H NMR (CDCl₃): δ 8.4 (s, 1H), 7.9 (m, 1H), 7.5 (m, 2H), 7.2 (m,2H), 4.75 (m, 3H), 4.6 (m, 1H), 4.45 (m, 1H), 3.9 (m, 4H), 3.4 (m, 1H),2.7 (s, 3H), 1.05 (s, 9H), 0.4 (br s, 6H); ESMS m/z 627 (M-H)⁻.

176: ¹H NMR (CDCl₃): δ 8.4 (s, 1H), 7.9 (m, 1H), 7.5 (m, 2H), 7.1 (m,2H), 4.7 (m, 3H), 4.6 (m, 1H), 4.45 (m, 1H), 3.9 (m, 4H), 3.4 (m, 1H),2.7 (s, 3H), 1.05 (s, 9H), 0.4 (br s, 6H).

94: ¹H NMR (CDCl₃): δ 8.5 (d, 2H), 8.2 (d, 2H), 7.9 (s, 2H), 7.8 (d,2H), 7.5 (m, 2H), 7.4 (m, 4H), 6.8 (d, 2H), 4.7 (d, 1H), 4.5 (m, 1H),4.4 (m, 1H), 3.9 (m, 6H), 3.3 (m, 1H), 2.9 (t, 2H), 2.7 (s, 3H), 2.5 (s,3H), 2.1 (m, 2H), 1.6 (s, 6H), 1.0 (s, 9H), 0.3 (br s, 6H).

ESMS m/z 1038 (M-H)⁻.

Example 2 2.1 Synthesis Methodology

2.1a Synthesis of Compound 1

The compounds of Formula I are readily prepared by reacting theappropriate spirocyclopropylcyclohexadienyl analog (Compounds B) withthe activated heterocyclic compounds A using sodium hydride inN,B-dimethylformamide (DMF) or tetrahydrofuran (THF). The resultingcompound 28 is then converted to compound 29 by treatment with theappropriate halo-acid, such as hydrochloric acid. Coupling with compound29 by in situ activation is used to produce compound 1, a compound ofFormula I.

Other compounds of Formula I are prepared according to publishedprocedures, which are modified to make additional analogs usingprocedures well known to those skilled in the art, such as reductions,oxidations, additions, aqueous extractions, evaporation, andpurification.

2.1b Synthesis of Compound A

To a solution of 5-nitro-2-carboxylic acid (0.83 g, 4.0 mmol) inN,N-dimethylformamide (60 mL) at 0° C. was added EDC (1.15 g, 6.0 mmol).The resulting suspension was stirred at 0° C. for 45 min, by which timethe EDC had completely dissolved. 4-Nitrophenol (0.83 g, 6.0 mmol) andDMAP (0.73 g, 6.0 mmol) were added and the resulting mixture stirred atambient temperature. After 13 hours, the mixture was diluted with ethylacetate and washed with a 10% aqueous citric acid solution twice,followed by water, and brine, then dried over Na₂SO₄, filtered andconcentrated in vacuo. Purification of the resultant residue by flashcolumn chromatography on silica gel (7% ethyl acetate in methylenechloride) afforded 1.02 g (78%) of A as a yellow solid: ¹H NMR (CDCl₃) δ9.0 (br s, 1H), 8.2 (d, 2H), 7.8 (m, 2H), 7.4 (d, 2H), 7.3 (s, 1H), 6.8(s, 1H).

2.1c Synthesis of Compound 28

To a solution of B (20 mg, 0.08 mmol) in N,N-dimethylformamide (1.0 mL)at −40° C. was added a suspension of sodium hydride (4.0 mg, 0.1 mmol,60% in oil) in N,N-dimethylformamide (1.0 mL). The resulting mixture wasallowed to warm to 0° C. slowly (1.5 h), then cooled back to −40° C. A(37 mg, 0.1 mmol) was added and the mixture allowed to warm to 0° C.slowly (1.5 h) where it was kept for 20 min. The mixture was cooled to−30° C., quenched with acetic acid (10 μL), stirred for 10 min, dilutedwith ethyl acetate, then washed with water then brine. The organic layerwas separated and dried over MgSO₄, filtered and concentrated in vacuo.Purification by flash column chromatography on silica gel (50% to 100%ethyl acetate in methylene chloride) afforded 16.3 mg (43%) of 28 as aslightly yellow solid: ¹H NMR (CDCl₃) δ 11.3 (br s, 1H), 9.4 (br s, 1H),7.4 (m, 2H), 7.1 (s, 1H), 6.95 (s, 1H), 6.8 (s, 1H), 4.4 (s, 2H), 3.8(s, 3H), 3.8 (m, 1H), 2.6 (s, 3H), 2.4 (dd, 1H), 1.4 (m, 1H). ESMS m/z490 (M-H)⁻.

2.1d Synthesis of Compound 29

To a solution of 28 (50 mg, 0.103 mmol) in N,N-dimethylformamide (1.0mL) was treated with 1 mL of anhydrous hydrochloric acid (1.0 M indioxane). The resulting solution was stirred at ambient temperature for30 min, then concentrated of solvent. Purification of the resultingresidue by flash column chromatography on silica gel (50% to 100% ethylacetate in methylene chloride) afforded 50 mg (100%) of 29 as a slightlyyellow solid: ¹H NMR (CDCl₃) δ 11.3 (br s, 1H), 9.4 (br s, 1H), 7.4 (m,2H), 7.1 (s, 1H), 6.95 (s, 1H), 6.8 (s, 1H), 4.4 (s, 2H), 3.8 (s, 3H),3.8 (m, 1H), 2.6 (s, 3H), 2.4 (dd, 1H), 1.4 (m, 1H).

2.1e Synthesis of Compound 1

To a solution of 29 (66 mg, 0.136 mmol) in anhydrous methylene chloride(10 mL) at −70° C. was added 4-nitro-phenylchloroformate (55 mg, 0.273mmol), followed by triethylamine (27 mg, 0.273 mmol). The resultingmixture was allowed to warm slowly. After 2 hours, the mixture wasplaced in an ice bath and amine C (82 mg, 0.273 mmol) was added in oneportion. The resulting mixture was stirred at ambient temperatureovernight. After 22 hours, the mixture was poured into saturated aqueousNaHCO₃ (aq). The aqueous layer was separated and extracted withmethylene chloride. The combined organic extracts were dried over MgSO₄,filtered and concentrated in vacuo. Purification by flash columnchromatography on silica gel (1% to 2% methanol in methylene chloride)afforded 42 mg (38%) of 1 as a slightly yellow solid: ¹H NMR (CDCl₃) δ11.0 (s, 1H), 8.6 (s, 1H), 8.4 (d, 1H), 8.2 (br d, 1H), 7.6 (m, 2H), 6.8(m, 1H), 4.8 (m, 1H), 4.7 (m, 1H), 4.5 (m, 1H), 4.1 (m, 2H), 3.9 (s,3H), 3.4 (m, 5H), 2.7 (d, 3H), 1.6 (s, 6H), 1.4 (s, 9H), 1.2 (m, 4H),0.9 (m, 9H).

In a similar manner the following compounds were prepared:

220: ¹H NMR (CDCl₃): δ 11.4 (d, 1H), 8.7 (s, 1H), 8.4 (, dd, 1H), 8.1(br s, 1H), 7.75 (d, 1H), 7.65 (s, 1H), 5.8 (br s, 2H), 4.7 (m, 1H), 4.5(m, 2H), 4.2 (m, 1H), 3.9 (s, 3H), 3.4 (m, 1H), 3.0 (s, 2H), 2.75 (s,3H), 2.65 (s, 3H).

ESMS m/z 613 (M-H)⁻.

222: ¹H NMR (CDCl₃): δ 10.3 (s, 1H), 8.7 (d, 2H), 8.4 (d, 1H), 8.1 (m,1H), 7.7 (d, 2H), 7.6 (m, 1H), 6.9 (d, 2H), 4.9 (d, 1H), 4.7 (m, 1H),4.5 (m, 3H), 3.95 (s, 3H), 3.85 (s, 3H), 3.6 (m, 1H), 3.4 (m, 1H), 3.1(s, 3H), 2.7 (s, 3H), 2.3 (s, 3H).

ESMS m/z 745 (M-H)⁻.

224: ESMS m/z 899 (M-H)⁻.

229: ESMS m/z 598 (M-H)⁻.

233: ESMS m/z 816 (M-H)⁻.

235: ESMS m/z 913 (M-H)⁻.

8: ESMS m/z 856 (M-H)⁻.

232: ESMS m/z 612 (M-H)⁻.

234: ESMS m/z 952 (M-H)⁻.

Example 3 3.1 Synthesis Methodology

3.1a Synthesis of Compound 239

The compounds of Formula I are readily prepared by reacting theappropriate spirocyclopropylcyclohexadienyl analog (Compounds B) withthe activated heterocyclic compounds A using sodium hydride inN,B-dimethylformamide (DMF) or tetrahydrofuran (THF). The resultingcompound 28 is then converted to compound 29 by treatment with theappropriate halo-acid, such as hydrochloric acid. Compound 29 activatedas the 4-nitrophenylester and coupled with compound C to give compound239, a compound of Formula I.

Other compounds of Formula I are prepared according to publishedprocedures, which are modified to make additional analogs usingprocedures well known to those skilled in the art, such as reductions,oxidations, additions, aqueous extractions, evaporation, andpurification.

3.1 b Synthesis of Compound A

To a solution of 5-nitro-2-carboxylic acid (0.83 g, 4.0 mmol) inN,N-dimethylformamide (60 mL) at 0° C. was added EDC (1.15 g, 6.0 mmol).The resulting suspension was stirred at 0° C. for 45 min, by which timethe EDC had completely dissolved. 4-Nitrophenol (0.83 g, 6.0 mmol) andDMAP (0.73 g, 6.0 mmol) were added and the resulting mixture stirred atambient temperature. After 13 hours, the mixture was diluted with ethylacetate and washed with a 10% aqueous citric acid solution twice,followed by water, and brine, then dried over Na₂SO₄, filtered andconcentrated in vacuo. Purification of the resultant residue by flashcolumn chromatography on silica gel (7% ethyl acetate in methylenechloride) afforded 1.02 g (78%) of A as a yellow solid: ¹H NMR (CDCl₃) δ9.0 (br s, 1H), 8.2 (d, 2H), 7.8 (m, 2H), 7.4 (d, 2H), 7.3 (s, 1H), 6.8(s, 1H).

3.1c Synthesis of Compound 28

To a solution of B (20 mg, 0.08 mmol) in N,N-dimethylformamide (1.0 mL)at −40° C. was added a suspension of sodium hydride (4.0 mg, 0.1 mmol,60% in oil) in N,N-dimethylformamide (1.0 mL). The resulting mixture wasallowed to warm to 0° C. slowly (1.5 h), then cooled back to −40° C. A(37 mg, 0.1 mmol) was added and the mixture allowed to warm to 0° C.slowly (1.5 h) where it was kept for 20 min. The mixture was cooled to−30° C., quenched with acetic acid (10 μL), stirred for 10 min, dilutedwith ethyl acetate, then washed with water then brine. The organic layerwas separated and dried over MgSO₄, filtered and concentrated in vacuo.Purification by flash column chromatography on silica gel (50% to 100%ethyl acetate in methylene chloride) afforded 16.3 mg (43%) of 28 as aslightly yellow solid: ¹H NMR (CDCl₃) δ 11.3 (br s, 1H), 9.4 (br s, 1H),7.4 (m, 2H), 7.1 (s, 1H), 6.95 (s, 1H), 6.8 (s, 1H), 4.4 (s, 2H), 3.8(s, 3H), 3.8 (m, 1H), 2.6 (s, 3H), 2.4 (dd, 1H), 1.4 (m, 1H). ESMS m/z490 (M-H)—.

3.1d Synthesis of Compound 29

To a solution of 28 (50 mg, 0.103 mmol) in N,N-dimethylformamide (1.0mL) was treated with 1 mL of anhydrous hydrochloric acid (1.0 M indioxane). The resulting solution was stirred at ambient temperature for30 min, then concentrated of solvent. Purification of the resultingresidue by flash column chromatography on silica gel (50% to 100% ethylacetate in methylene chloride) afforded 50 mg (100%) of 29 as a slightlyyellow solid: ¹H NMR (CDCl₃) δ 11.3 (br s, 1H), 9.4 (br s, 1H), 7.4 (m,2H), 7.1 (s, 1H), 6.95 (s, 1H), 6.8 (s, 1H), 4.4 (s, 2H), 3.8 (s, 3H),3.8 (m, 1H), 2.6 (s, 3H), 2.4 (dd, 1H), 1.4 (m, 1H).

3.1e Synthesis of Compound 239

To a suspension of 29 (24 mg, 0.05 mmol) in methylene chloride (5 ml) at−78° C. was added 4-nitrophenyl chloroformate (40 mg, 0.2 mmol), andtriethylamine (28 μl, 0.2 mmol). The reaction mixture was allowed towarm to room temperature, then concentrated in vacuo. The resultingresidue was washed with diethyl ether, then dried over vacuum to give 9as a yellow solid. Yellow solid 9 (19 mg, 0.029 mmol) in was dissolvedin methylene chloride (3 ml) and the amine C (20 mg, 0.029 mmol) wasadded, followed by triethylamine (8.3 μl, 0.06 mmol). The reaction wasstirred for 16 hours then concentrated in vacuo. The resulting residuewas purified by flash chromatography using silica gel and 40:1 methylenechloride:methanol to give 12 mg (45% yield) of 239 as a yellow solid: ¹HNMR (CDCl₃): δ 9.9 (s, 1H), 9.7 (s, 1H), 8.7 (s, 1H), 8.4 (dd, 1H), 8.3(d, 1H), 8.1 (br s, 1H), 7.75 (d, 1H), 7.65 (s, 1H), 6.7 (m, 2H), 4.75(m, 1H), 4.55 (m, 2H), 3.9 (m, 4H), 3.8 (m, 4H), 3.5 (m, 18H), 3.0 (m,2H), 2.7 (s, 3H), 2.5 (m, 4H), 1.7 (m, 2H), 1.4 (m, 10H), 1.0 (m, 2H).

ESMS m/z 1100 (M-H)⁻.

In a similar manner the following compounds were made:

238: ¹H NMR (CDCl₃): δ 10.3 (s, 1H), 8.7 (s, 1H), 8.5 (m, 1H), 8.4 (d,1H), 8.2 (br s, 1H), 7.7 (m, 4H), 7.2 (m, 1H), 4.8 (m, 1H), 4.6 (m, 2H),3.9 (m, 4H), 3.7 (m, 2H), 3.4 (m, 1H), 3.2 (m, 2H), 2.9 (s, 3H), 2.5 (s,3H).

ESMS m/z 710 (M-H)⁻.

242: ¹H NMR (CDCl₃): δ 9.7 (br s, 1H), 9.0 (br s, 1H), 8.6 (s, 1H), 8.4(d, 1H), 8.1 (br s, 1H), 7.7 (m, 1H), 7.5 (m, 1H), 7.4 (m, 1H), 4.7 (d,1H), 4.5 (m, 2H), 3.9 (s, 4H), 3.8 (m, 1H), 3.6 (m, 1H), 3.4 (m, 1H),3.0 (m, 5H), 2.6 (s, 3H), 2.5 (m, 2H), 1.5 (m, 9H), 1.4 (m, 6H).

244: ¹H NMR (CDCl₃): δ 8.6 (br s, 1H), 8.4 (d, 1H), 8.1 (m, 1H), 7.7 (m,6H), 6.9 (m, 2H), 4.75 (m, 1H), 4.55 (m, 2H), 4.1 (m, 2H), 3.9 (m, 4H),3.7 (m, 12H), 3.5 (m, 2H), 3.4 (m, 3H), 3.2 (m, 3H), 3.1 (m, 5H), 2.7(s, 6H), 2.1 (m, 2H), 1.5 (s, 6H).

248: ¹H NMR (CDCl₃): δ 9.9 (s, 1H), 9.3 (br s, 1H), 8.1 (m, 1H), 7.5 (m,3H), 7.1 (m, 2H), 4.8 (d, 1H), 4.5 (m, 2H), 3.95 (s, 3H), 3.85 (s, 3H),3.8 (m, 1H), 3.7 (m, 2H), 3.4 (m, 1H), 3.0 (m, 8H), 2.5 (m, 2H), 1.5(dd, 6H), 1.3 (s, 9H).

250: ¹H NMR (CDCl₃): δ 9.4 (s, 1H), 8.6 (s, 1H), 8.3 (m, 1H), 8.1 (m,1H), 7.9 (m, 2H), 7.5 (m, 7H), 7.1 (m, 4H), 4.8 (m, 1H), 4.5 (m, 2H),4.3 (m, 1H), 4.95 (s, 3H), 4.85 (s, 3H), 4.7 (m, 1H), 3.4 (m, 1H), 3.1(m, 4H), 2.7 (s, 3H), 2.2 (s, 3H), 1.5 (s, 6H).

272: ¹H NMR (DMSO): δ 12.0 (m, 1H), 8.8 (br s, 1H), 8.3 (m, 1H), 7.9 (m,3H), 7.7 (m, 3H), 6.9 (m, 2H), 4.85 (s, 1H), 4.7 (m, 1H), 4.5 (m, 3H),3.8 (m, 4H), 3.6 (m, 4H), 3.4 (m, 5H), 3.1 (m, 6H), 2.7 (s, 3H), 2.2 (m,2H), 1.4 (s, 6H).

Example 4 Proliferation Assays

The assay which was selected for measuring the biological activity ofthe cytotoxic compounds is the well established ³H-thymidineproliferation assay. This is a convenient method for quantitatingcellular proliferation as it evaluates DNA synthesis by measuring theincorporation of exogenous radiolabeled ³H-thymidine. This assay ishighly reproducible and can accommodate large numbers of compounds.

Promyelocytic leukemia cells, HL-60, were cultured in RPMI mediacontaining 10% heat inactivated fetal calf serum (FCS). On the day ofthe study, the cells were collected, washed and resuspended at aconcentration of 0.5×10⁶ cells/ml in RPMI containing 10% FCS. 100 μl ofcell suspension was added to 96 well plates. Serial dilutions (3-foldincrements) of doxorubicin or test compounds were made and 100 μl ofcompounds were added per well. Finally 10 μl of a 100 μCi/ml³H-thymidine was added per well and the plates were incubated for 24hours. The plates were harvested using a 96 well Harvester (PackardInstruments) and counted on a Packard Top Count counter. Four parameterlogistic curves were fitted to the ³H-thymidine incorporation as afunction of drug molarity using Prism software to determine IC₅₀ values.

The compounds of the invention generally have an IC₅₀ value in the aboveassay of from about 1 μM to about 100 nM, preferably from about 10 μM toabout 10 nM.

Each of the patent applications, patents, publications, and otherpublished documents mentioned or referred to in this specification isherein incorporated by reference in its entirety, to the same extent asif each individual patent application, patent, publication, and otherpublished document was specifically and individually indicated to beincorporated by reference.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention and the appended claims. In addition, many modifications maybe made to adapt a particular situation, material, composition ofmatter, process, process step or steps, to the objective, spirit andscope of the present invention. All such modifications are intended tobe within the scope of the claims appended hereto.

1. A compound having the structure:

wherein Z is NH; X is O; R³ is a member selected from the groupconsisting of (═O) and OR¹¹, wherein R¹¹ is a member selected from thegroup consisting of H, substituted or unsubstituted C₁₋₆ alkyl,substituted or unsubstituted C₁₋₆ heteroalkyl, C(O)R¹², C(O)OR¹²,C(O)NR¹²R¹³, in which R¹² and R¹³ are H or R¹² and R¹³ together with thenitrogen or carbon atom to which they are attached are joined to form asubstituted or unsubstituted heterocycloalkyl ring system having from 4to 6 members, optionally containing two or more heteroatoms; R⁵ is H; R⁴is NHR¹⁶, wherein R¹⁶ is

wherein X⁴ is an antibody or a reactive functional group having thestructure:

wherein

is a single or double bond; L³ is a linker selected from substituted orunsubstituted C₁₋₁₀ alkyl and substituted or unsubstituted C₁₋₁₀heteroalkyl groups; AA¹, AA^(c) and AA^(c+1) are members independentlyselected from natural and unnatural α-amino acids; L⁴ is a linkerselected from substituted or unsubstituted C₁₋₁₀ alkyl, substituted orunsubstituted C₁₋₁₀ heteroalkyl groups, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, and substituted orunsubstituted cycloalkyl; p and t are integers independently selectedfrom 0 and 1; and c is an integer from 0 to 5; and R⁶ is a single bondwhich is either present or absent and when present R⁶ and R⁷ are joinedto form a cyclopropyl ring; and R⁷ is CH₂—XI or —CH₂— joined in saidcyclopropyl ring with R⁶, wherein X¹ is a leaving group, or apharmaceutically acceptable salt thereof.
 2. The compound of claim 1,wherein R⁶ is absent, R⁷ is CH₂—X¹, R³ is OR¹¹, and R¹¹ is C(O)NR¹²R¹³,in which R¹² and R¹³ together with the nitrogen atom to which they areattached are joined to form a substituted or unsubstitutedheterocycloalkyl ring system having from 4 to 6 members, optionallycontaining two or more heteroatoms.
 3. The compound of claim 1, whereinL³ is a substituted or unsubstituted C₁₋₁₀ heteroalkyl group and L⁴ issubstituted or unsubstituted aryl or heteroaryl.
 4. The compound ofclaim 1, wherein p is 1, t is 1, and c is 0 or
 1. 5. The compound ofclaim 1, wherein AA¹ is valine.
 6. The compound of claim 1, wherein X⁴is an antibody.
 7. The compound of claim 1, wherein R¹ is absent, R⁷ isCH₂—X¹, R³ is OR¹¹, and R¹¹ is C(O)NR¹²R¹³, in which R¹² and R¹³together with the nitrogen atom to which they are attached are joined toform a substituted or unsubstituted heterocycloalkyl ring system havingfrom 4 to 6 members, containing two or more heteroatoms; p is 1 and L³is a substituted or unsubstituted C₁₋₁₀ heteroalkyl; and t is 1 and L⁴is substituted or unsubstituted aryl or heteroaryl.
 8. The compound ofclaim 7, wherein c is 0 or 1 and AA¹ is valine.
 9. The compound of claim7, wherein X⁴ is an antibody.
 10. The compound of claim 1, wherein R⁶ isabsent, R⁷ is CH₂—X¹, R³ is OR¹¹, and X¹ is halogen.
 11. A compoundhaving the structure:

wherein A is a member selected from substituted or unsubstituted phenyland substituted or unsubstituted pyrrole; Z is a member selected from Oand NR²³; X is a member selected from O and NR²³; wherein each R²³ isindependently a member selected from H, substituted or unsubstitutedC₁₋₁₀ alkyl, substituted or unsubstituted C₁₋₁₀ heteroalkyl, and acyl;R³ is a member selected from the group consisting of (═O) and OR¹¹,wherein R¹¹ is a member selected from the group consisting of H,substituted or unsubstituted C₁₋₁₀ alkyl, substituted or unsubstitutedC₁₋₁₀ heteroalkyl, diphosphates, triphosphates, acyl, C(O)R¹², C(O)OR¹²,C(O)NR¹²R¹³, P(O)(OR¹²)₂, C(O)CHR¹²R¹³, SR¹² and SiR¹²R¹³R¹⁴, in whichR¹², R¹³, and R¹⁴ are members independently selected from H, substitutedor unsubstituted C₁₋₁₀ alkyl, substituted or unsubstituted C₁₋₁₀heteroalkyl and substituted or unsubstituted aryl, wherein R¹² and R¹³together with the nitrogen or carbon atom to which they are attached areoptionally joined to form a substituted or unsubstitutedheterocycloalkyl ring system having from 4 to 6 members, optionallycontaining two or more heteroatoms; R⁴ is H, substituted orunsubstituted C₁₋₁₀ alkyl, halogen, or NO₂; R⁵ is NR¹⁵R¹⁶, wherein R¹⁵is H, substituted or unsubstituted C₁₋₁₀ alkyl, or substituted orunsubstituted C₁₋₁₀ heteroalkyl; R¹⁶ is

wherein X⁴ is member selected from protected or unprotected reactivefunctional groups, detectable labels and targeting agents; L³ is alinker selected from substituted or unsubstituted C₁₋₂₄ alkyl andsubstituted or unsubstituted C₁₋₂₄ heteroalkyl groups; AA¹, AA^(c) andAA^(c+1) are members independently selected from natural and unnaturalα-amino acids; L⁴ is a linker selected from substituted or unsubstitutedC₁₋₂₄ alkyl, substituted or unsubstituted C₁₋₂₄ heteroalkyl groups,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, and substituted or unsubstituted cycloalkyl; p and t areintegers independently selected from 0 and 1; and c is an integer from 0to 20; and R⁶ is a single bond which is either present or absent andwhen present R⁶ and R⁷ are joined to form a cyclopropyl ring; and R⁷ isCH₂—XI or —CH₂— joined in said cyclopropyl ring with R⁶, wherein X¹ is aleaving group; or a pharmaceutically acceptable salt thereof.
 12. Thecompound of claim 11 wherein Z is a member selected from O and NH; X isO; R⁶ is absent; and R⁷ is CH₂-halogen.
 13. The compound of claim 11wherein R⁶ is absent, R⁷ is CH₂—X¹, R³ is OR¹¹, and R¹¹ is a memberselected from the group consisting of H, substituted or unsubstitutedC₁₋₆ alkyl, substituted or unsubstituted C₁₋₆ heteroalkyl, C(O)R¹²,C(O)OR¹², C(O)NR¹²R¹³, in which R¹² and R¹³ are H or R¹² and R¹³together with the nitrogen or carbon atom to which they are attached arejoined to form a substituted or unsubstituted heterocycloalkyl ringsystem having from 4 to 6 members, optionally containing two or moreheteroatoms.
 14. The compound of claim 13 wherein R⁶ is absent, R⁷ isCH₂—X¹, R³ is OR¹¹, and R¹¹ is a substituted or unsubstituted C₁₀heteroalkyl or C(O)NR¹²R¹³.
 15. The compound of claim 14 wherein R⁶ isabsent, R⁷ is CH₂—X¹, R³ is OR¹¹, and R¹¹ is C(O)NR¹²R¹³ and wherein R¹²and R¹³ together with the nitrogen or carbon atom to which they areattached are joined to form a substituted or unsubstitutedheterocycloalkyl ring system having from 4 to 6 members, optionallycontaining two or more heteroatoms.
 16. The compound of claim 11,wherein X⁴ is an antibody or a reactive functional group having thestructure:

where

is a single or double bond.
 17. The compound of claim 11, wherein L³ isa linker selected from substituted or unsubstituted C₁₋₁₀ alkyl andsubstituted or unsubstituted C₁₋₁₀ heteroalkyl groups; AA¹, AA^(c) andAA^(c+1) are members independently selected from natural and unnaturalα-amino acids; L⁴ is a linker selected from substituted or unsubstitutedC₁₋₁₀ alkyl, substituted or unsubstituted C₁₋₁₀ heteroalkyl groups, andsubstituted or unsubstituted aryl, p and t are both 1; and c is aninteger from 0 to
 5. 18. The compound of claim 11, wherein L³ is asubstituted or unsubstituted C₁₋₁₀ heteroalkyl group and L⁴ issubstituted or unsubstituted aryl or heteroaryl.
 19. The compound ofclaim 11, wherein p is 1, t is 1, and c is 0 or
 1. 20. The compound ofclaim 11, wherein AA¹ is valine.
 21. The compound of claim 20, whereinX⁴ is an antibody.
 22. A method of reducing the toxicity of a cytotoxiccompound, the method comprising: preparing a linked conjugate by linkinga compound having the structure I:

wherein A is a member selected from substituted or unsubstituted phenyland substituted or unsubstituted pyrrole; Z is a member selected from O,S and NR²³; X is a member selected from O, S and NR²³; wherein each R²³is independently a member selected from H, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, and acyl; R³ is amember selected from the group consisting of (═O) and OR¹¹, wherein R¹¹is a member selected from the group consisting of H, substituted alkyl,unsubstituted alkyl, substituted heteroalkyl, unsubstituted heteroalkyl,diphosphates, triphosphates, acyl, C(O)R¹², C(O)OR¹², C(O)NR¹²R¹³,P(O)(OR¹²)₂, C(O)CHR¹²R¹³, SR¹² and SiR¹²R¹³R¹⁴, in which R¹², R¹³, andR¹⁴ are members independently selected from H, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl, wherein R¹² and R¹³ together with thenitrogen or carbon atom to which they are attached are optionally joinedto form a substituted or unsubstituted heterocycloalkyl ring systemhaving from 4 to 6 members, optionally containing two or moreheteroatoms; R⁴ and R⁵ are members independently selected from H,substituted or unsubstituted alkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl, halogen, NO₂, NR¹⁵R¹⁶, NC(O)R¹⁵, OC(O)NR¹⁵R¹⁶,OC(O)OR¹⁵, C(O)R¹⁵, SR¹⁵, and OR¹⁵ wherein R¹⁵ and R¹⁶ are independentlyselected from H, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocycloalkyl and substituted or unsubstituted peptidyl; R⁶ is asingle bond which is either present or absent and when present R⁶ and R⁷are joined to form a cyclopropyl ring; and R⁷ is CH₂—X¹ or CH₂— joinedin said cyclopropyl ring with R⁶, wherein X¹ is a leaving group, throughthe R⁴, R¹⁵, or R¹⁶ moiety to a peptidyl moiety comprising:

wherein X⁴ is member selected from protected or unprotected reactivefunctional groups, detectable labels and targeting agents; L³ is alinker selected from substituted or unsubstituted alkyl and substitutedor unsubstituted heteroalkyl groups; AA¹, AA^(c)and AA^(c+1) are membersindependently selected from natural and unnatural α-amino acids; L⁴ is alinker selected from substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl groups, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, and substituted orunsubstituted cycloalkyl; p and t are integers independently selectedfrom 0 and 1; and c is an integer from 0 to 20, wherein the linkedconjugate has reduced toxicity compared to a cytotoxic compound havingthe structure II:

wherein A, X, and Z of structures (I) and (II) are identical and R^(4′)and R^(5′) of structure (II) are identical to R⁴ and R⁵ of structure(I), respectively, except that the peptidyl moiety is replaced by H. 23.The method of claim 22, wherein said linked conjugate, when tested inhuman blood at 37° C. for 2 hours, has less than 20% cleavage by enzymespresent in the human blood.
 24. The method of claim 22, wherein R¹⁵ is Hand R¹⁶ links the compound to the peptidyl moiety.
 25. The method ofclaim 22, wherein Z is a member selected from O and NH; X is O; R⁶ isabsent; and R⁷ is CH₂-halogen.
 26. The method of claim 22, wherein R⁶ isabsent, R⁷ is CH₂—X¹, R³ is OR¹¹, and R¹¹ is a substituted orunsubstituted C₁₋₁₀ heteroalkyl or C(O)NR¹²R¹³.
 27. The method of claim26, wherein R⁶ is absent, R⁷ is CH₂—X¹, R³ is OR¹¹, and R¹¹ isC(O)NR¹²R¹³ and wherein R¹² and R¹³ together with the nitrogen or carbonatom to which they are attached are joined to form a substituted orunsubstituted heterocycloalkyl ring system having from 4 to 6 members,optionally containing two or more heteroatoms.
 28. The method of claim22, wherein X⁴ is an antibody or a reactive functional group having thestructure:

where

is a single or double bond.
 29. The method of claim 22, wherein L³ is alinker selected from substituted or unsubstituted C₁₋₁₀ alkyl andsubstituted or unsubstituted C₁₋₁₀ heteroalkyl groups; AA¹, AA^(c) andAA^(c+1) are members independently selected from natural and unnaturalα-amino acids; L⁴ is a linker selected from substituted or unsubstitutedC₁₋₁₀ alkyl, substituted or unsubstituted C₁₋₁₀ heteroalkyl groups, andsubstituted or unsubstituted aryl; p and t are both 1; and c is aninteger from 0 to
 5. 30. The method of claim 22, wherein L³ is asubstituted or unsubstituted C₁₋₆ heteroalkyl group and L⁴ issubstituted or unsubstituted aryl or heteroaryl.
 31. The method of claim22, wherein p is 1, t is 1, and c is 0 or
 1. 32. The method of claim 22,wherein AA¹ is valine.